Humanized neuraminidase antibody and methods of use thereof

ABSTRACT

Antibodies against influenza neuraminidase, compositions containing the antibodies, and methods of using the antibodies are provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/121,235, filed Sep. 6, 2011, now U.S. Pat. No. 8,734,803, which is aNational Stage application under 35 U.S.C. 371 of InternationalApplication No. PCT/US2009/058640, having an International Filing Dateof Sep. 28, 2009, which claims benefit of priority from U.S. ProvisionalApplication Ser. No. 61/100,740, filed Sep. 28, 2008, which isincorporated herein by reference in its entirety.

TECHNICAL FIELD

This document relates to antibodies against influenza, and moreparticularly to an influenza N1 neuraminidase-specific monoclonalantibody that can protect animals against live challenge with homologousH5N1 virus.

BACKGROUND

Influenza has a long history characterized by waves of pandemics,epidemics, resurgences and outbreaks. Influenza is a highly contagiousdisease with the potential to be devastating both in developing anddeveloped countries. In spite of annual vaccination efforts, influenzainfections result in substantial morbidity and mortality each year.Although pandemics do not occur very often, flu strains have recentlyemerged that increase the potential for an influenza pandemic. Anexample is the avian influenza virus of the type H5N1, which as causedan epidemic in poultry in Asia as well as in regions of Eastern Europe,and has persistently spread throughout the globe. The rapid spread ofinfection and the cross species transmission from birds to humans hasincreased the potential for outbreaks in human populations and the riskof a pandemic. The virus is highly pathogenic, with a mortality rate ofover fifty percent in birds as well as the few human cases that havebeen identified. Human to human transmission of the virus would have thepotential to result in rapid, widespread illness and mortality.

The major defense against influenza is vaccination. Influenza virusesare segmented, negative-strand RNA viruses belonging to the familyOrthomyxoviridae. Influenza virus hemagglutinin glycoprotein (HA)generally is considered the most important viral antigen with regard tothe stimulation of neutralizing antibodies and vaccine design. Thepresence of viral neuraminidase (NA) has been shown to be important forgenerating multi-arm protective immune responses against the virus.Antiviral agents that inhibit neuraminidase activity have been developedand can be an additional antiviral treatment upon infection. A thirdcomponent considered useful in the development of influenza antiviralsand vaccines is the ion channel protein M2.

Subtypes of the influenza virus are designated by different HA and NAthat are the result of antigenic shift. Furthermore, new strains of thesame subtype result from antigenic drift or from mutations in the HA orNA molecules that generate new and different epitopes. Although 15antigenic subtypes of HA have been documented, only three of thesesubtypes (H1, H2, and H3) have circulated extensively in humans.Vaccination has become paramount in the quest for improved quality oflife in both industrialized and underdeveloped nations. The majority ofavailable vaccines still follow the basic principles of mimickingaspects of infection in order to induce an immune response that couldprotect against the relevant infection. However, generation ofattenuated viruses of various subtypes and combinations can be timeconsuming and expensive. Along with emerging new technologies, in-depthunderstanding of a pathogen's molecular biology, pathogenesis, andinteractions with an individual's immune system has resulted in newapproaches to vaccine development and vaccine delivery. Thus, whiletechnological advances have improved the ability to produce improvedinfluenza antigens vaccine compositions, there remains a need to provideadditional sources of protection against to address emerging subtypesand strains of influenza.

SUMMARY

This document relates to antibody compositions and methods for producingantibody compositions, including production in plant systems. Thisdocument further relates to vectors encoding antibodies or antigenbinding fragments thereof, as well as fusion proteins, plant cells,plants, compositions, and kits comprising antibodies or antigen bindingfragments thereof, and therapeutic and diagnostic uses in associationwith influenza infection in a subject.

This document is based in part on the identification of an anti-H5N1neuraminidase monoclonal antibody that specifically inhibits N1neuraminidase activity of highly pathogenic avian influenza (HPAI)strains from clades 1, 2, and 3. The N1NA-specific mAb, 2B9, can inhibitenzymatic activity of NA from several strains of H5N1, includingoseltamivir-resistant HPAI isolates. The protective efficacy of thisantibody has been demonstrated in animal challenge models (e.g., mousemodels) using homologous virus. The specific and effective inhibition ofNINA renders this mAb a useful therapeutic tool in the treatment and/orprevention of human infection. The 2B9 mAb also can be useful fortreating and/or preventing infection with drug-(e.g., oseltamivir-and/or zanimivir-) resistant strains of HPAI. In addition, the mAb canbe a useful diagnostic tool for typing suspected H5N1 human isolates inconjunction with other diagnostic approaches.

Thus, this document provides antibodies against influenza neuraminidaseantigens, as well as antibody components produced in plants. Theantibodies can inhibit neuraminidase activity. Also provided areantibody compositions that are reactive against influenza neuraminidaseantigen. In addition, methods for production and use of the antibodiesand compositions are provided herein.

In one aspect, this document features an isolated monoclonal antibodythat binds neuraminidase, wherein the antibody has the ability toinhibit neuraminidase enzyme activity, and wherein the antibodycomprises a light chain variable region amino acid sequence as set forthin amino acids 1 to 127 of SEQ ID NO:5, and a heavy chain variableregion amino acid sequence as set forth in amino acids 1 to 137 of SEQID NO:6. The antibody can be an antigen-binding fragment of an antibody(e.g., an scFv, Fv, Fab′, Fab, diabody, linear antibody or F(ab′)₂antigen-binding fragment of an antibody, or a CDR, univalent fragment,single domain antibody). The antibody can be a human, humanized orpart-human antibody or antigen-binding fragment thereof (e.g., ahumanized antibody comprising a heavy chain amino acid sequence setforth in SEQ ID NO:7, a humanized antibody comprising a heavy chainamino acid sequence set forth in SEQ ID NO:8, a humanized antibodycomprising a light chain amino acid sequence set forth in SEQ ID NO:9,or a humanized antibody comprising a light chain amino acid sequence setforth in SEQ ID NO:10). The antibody can be a recombinant antibody.

In another aspect, this document features an antibody that bindsneuraminidase, wherein the antibody has the ability to inhibitneuraminidase enzyme activity, and wherein the antibody comprises alight chain amino acid sequence that is at least 85 percent identical(e.g., at least 90 percent identical, or at least 95 percent identical)to the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:10, anda heavy chain amino acid sequence that is at least 85 percent identical(e.g., at least 90 percent identical, or at least 95 percent identical)to the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:8.

The antibodies provided herein can be produced in a plant. Theantibodies can be operatively attached to a biological agent or adiagnostic agent (e.g., an agent that cleaves a substantially inactiveprodrug to release a substantially active drug, such as ananti-influenza agent, or an anti-viral agent such as an anti-influenzaagent). The antibodies can be operatively attached to a diagnostic,imaging or detectable agent (e.g., an X-ray detectable compound, aradioactive ion or a nuclear magnetic spin-resonance isotope, such as(a) the X-ray detectable compound bismuth (III), gold (III), lanthanum(III) or lead (II); (b) the detectable radioactive ion copper⁶⁷,gallium⁶⁷, gallium⁶⁸, indium¹¹³, iodine¹²³, iodine¹²⁵, iodine1³¹,mercury¹⁹⁷, mercury²⁰³, rhenium¹⁸⁶, rhenium¹⁸⁸, rubidium⁹⁷, rubidium¹⁰³,technetium^(99m) or yttrium⁹⁰; or (c) the detectable nuclear magneticspin-resonance isotope cobalt (II), copper (II), chromium (III),dysprosium (III), erbium (III), gadolinium (III), holmium (III), iron(II), iron (III), manganese (II), neodymium (III), nickel (II), samarium(III), terbium (III), vanadium (II) or ytterbium (III). The antibodiescan be operatively attached to biotin, avidin or to an enzyme thatgenerates a colored product upon contact with a chromogenic substrate.The antibodies can be operatively attached to the biological agent as afusion protein prepared by expressing a recombinant vector thatcomprises, in the same reading frame, a DNA segment encoding theantibody operatively linked to a DNA segment encoding the biologicalagent. The antibodies can be operatively attached to the biologicalagent via a biologically releasable bond or selectively cleavablelinker.

In another aspect, this document features a recombinant, plant-producedmonoclonal antibody that binds neuraminidase, wherein the antibody hasthe ability to inhibit neuraminidase enzyme activity, and wherein theantibody comprises a light chain amino acid sequence as set forth in SEQID NO:5, and a heavy chain amino acid sequence as set forth in SEQ IDNO:6.

This document also features a pharmaceutical composition comprising anantibody as described herein, and a pharmaceutically acceptable carrier.The composition can be formulated for parenteral administration. Theantibody can be a recombinant, plant-produced antibody. Thepharmaceutically acceptable composition can be an encapsulated orliposomal formulation. The composition can further comprise a secondtherapeutic agent.

Also provided herein is a method for treating an influenza infection ina subject in need thereof, comprising administering to the subject anamount of a composition as provided herein that is effective to reducesymptoms of the influenza infection in the subject.

In addition, this document features use of an antibody as describedherein for diagnosing a condition due to infection by a human influenzavirus, or for typing a human influenza virus, wherein binding of theantibody to the influenza virus is indicative of an N1 virus.

In still another aspect, this document features a method for treating asubject in need thereof, comprising providing a biological sample fromthe subject, contacting the biological sample with an antibody asprovided herein, and, if the antibody shows detectable binding to thebiological sample, administering the antibody to the subject. Thesubject can be a human patient (e.g., a human patient diagnosed ashaving influenza, and in some cases a human patient diagnosed as havingan oseltamivir-resistant strain of influenza).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a depiction of a plasmid construct used to expressneuraminidase in plants.

FIG. 2 is a graph plotting results of an ELISA, indicating differentialbinding of the 2B9 anti-N1 neuraminidase monoclonal antibody to wellscoated with NIBRG-14 virions, NINA protein, N2NA protein, and H5HAprotein, as indicated.

FIG. 3 is a graph plotting inhibition of N1 neuraminidase activity by2B9 or, as a control, anti-RSV F protein, using the molar ratios asindicated on the X axis.

FIG. 4 is a graph plotting percent survival of mice treated with 2B9 orPBS prior to challenge with the A/VN/1203/04 H5N1 influenza strain.

FIG. 5 is a picture of a gel stained with Coomassie blue dye, showingthe light (lower bands) and heavy (upper bands) chains of humanized,plant-produced 2B9 (h2B9) antibodies.

FIG. 6 is a graph plotting antigen binding by the indicatedconcentrations of various humanized 2B9 antibodies, as determined by anELISA.

FIG. 7 is a series of graphs plotting the half life ofhybridoma-produced (top panels) or plant-produced (bottom panels) 2B9antibody, administered to mice intravenously (left panels) orintramuscularly (right panels).

DETAILED DESCRIPTION

Influenza Antigens

Influenza antigen proteins can include any immunogenic protein orpeptide capable of eliciting an immune response against influenza virus.Generally, immunogenic proteins of interest include influenza antigens(e.g., influenza proteins), immunogenic portions thereof, or immunogenicvariants thereof and combinations of any of the foregoing.

Influenza antigens can include full-length influenza proteins orfragments of influenza proteins. Where fragments of influenza proteinsare utilized, such fragments can retain immunological activity (e.g.,cross-reactivity with anti-influenza antibodies). Hemagglutinin andneuraminidase have the capacity to induce immunoprotective responsesagainst viral infection, and are primary antigens of interest ingenerating antibodies.

Amino acid sequences of a variety of different influenza NA proteins(e.g., from different subtypes, or strains or isolates) are known in theart and are available in public databases such as GenBank. Exemplaryfull length protein sequences for NA of two influenza subtypes areprovided below. The italicized portion at the beginning of each sequencerepresents the anchor peptide for that protein.

Vietnam H5N1 NA (NAV): (SEQ ID NO: 1)MNPNQKIITIGSICMVTGIVSLMLQIGNMISIWVSHSIHTGNQHQSEPISNTNLLTEKAVASVKLAGNSSLCPINGWAVYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLNDKHSNGTVKDRSPHRTLMSCPVGEAPSPYNSRFESVAWSASACHDGTSWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTVMTDGPSNGQASHKIFKMEKGKVVKSVELDAPNYHYEECSCYPDAGEITCVCRDNWHGSNRPWVSFNQNLEYQIGYICSGVFGDNPRPNDGTGSCGPVSSNGAGGVKGFSFKYGNGVWIGRTKSTNSRSGFEMIWDPNGWTETDSSFSVKQDIVAITDWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPKESTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK  Wyoming H3N2 NA (NAW):(SEQ ID NO: 2) MNPNQKIITIGSVSLTISTICFFMQIAILITTVTLHFKQYEFNSPPNNQVMLCEPTIIERNITEIVYLTNTTIEKEICPKLAEYRNWSKPQCNITGFAPFSKDNSIRLSAGGDIWVTREPYVSCDPDKCYQFALGQGTTLNNVHSNDTVHDRTPYRTLLMNELGVPFHLGTKQVCIAWSSSSCHDGKAWLHVCVTGDDENATASFIYNGRLVDSIVSWSKKILRTQESECVCINGTCTVVMTDGSASGKADTKILFIEEGKIVHTSTLSGSAQHVEECSCYPRYPGVRCVCRDNWKGSNRPIVDINIKDYSIVSSYVCSGLVGDTPRKNDSSSSSHCLDPNNEEGGHGVKGWAFDDGNDVWMGRTISEKLRSGYETFKVIEGWSNPNSKLQINRQVIVDRGNRSGYSGIFSVEGKSCINRCFYVELIRGRKQETEVLWTSNSIVVFCGTS  GTYGTGSWPDGADINLMPI

While sequences of exemplary influenza antigens are provided herein, anddomains depicted for NA have been provided for exemplary strains, itwill be appreciated that any sequence having immunogenic characteristicsof a domain of NA can alternatively be employed. One skilled in the artwill readily be capable of generating sequences having at least 75%,80%, 85%, 90%, 95%, or more than 95% identity to the provided antigens.In certain embodiments, influenza antigens can be polypeptides having atleast 95%, 96%, 97%, 98%, or more identity to a domain NA, or a portionof a domain NA, wherein the polypeptide retains immunogenic activity.Percent sequence identity is determined as described below. Sequenceshaving sufficient identity to influenza antigen(s) that retainimmunogenic characteristics can be capable of binding with antibodiesthat react with domains (antigen(s)) provided herein Immunogeniccharacteristics often include three dimensional presentation of relevantamino acids or side groups. One skilled in the art can readily identifysequences with modest differences in sequence (e.g., with difference inboundaries and/or some sequence alternatives, that, nonetheless preserveimmunogenic characteristics). Further, one will appreciate that anydomains, partial domains or regions of amino acid sequence of influenzaantigen (e.g., NA) which are immunogenic can be generated usingconstructs and methods provided herein. Still further, domains orsubdomains can be combined, separately and/or consecutively forproduction of influenza antigens.

Sequences of particular neuraminidase subtypes have been used asexemplary antigens, as described in detail herein. Various subtypes ofinfluenza virus exist and continue to be identified as new subtypesemerge. It will be understood by one skilled in the art that the methodsand compositions provided herein can be adapted to utilize sequences ofadditional subtypes. Such variation is contemplated and encompassedwithin the methods and compositions provided herein.

Transgenic plants expressing influenza antigen(s) (e.g., influenzaprotein(s) or fragments thereof) can be used for production in plantsystems. Transgenic plants can be produced using methods well known inthe art to generate stable production crops, for example. Plantsutilizing transient expression systems also can be used for productionof influenza antigen(s). When utilizing plant expression systems,whether transgenic or transient expression in plants is utilized, any ofnuclear expression, chloroplast expression, mitochondrial expression, orviral expression can be used according to the applicability of thesystem to antigen desired. Furthermore, other expression systems forproduction of antigens can be used. For example, mammalian expressionsystems (e.g., mammalian cell lines such as CHO cells), bacterialexpression systems (e.g., E. coli), insect expression systems (e.g.,baculovirus), yeast expression systems, and in vitro expression systems(e.g., reticulate lysates) can be used to express antigens.

Production of Influenza Antigens:

Influenza antigens (including influenza protein(s), fragments, and/orvariants thereof) can be produced in any desirable system; production isnot limited to plant systems. Vector constructs and expression systemsare well known in the art and can be adapted to incorporate use ofinfluenza antigens provided herein. For example, influenza antigens(including fragments and/or variants) can be produced in knownexpression systems, including mammalian cell systems, transgenicanimals, microbial expression systems, insect cell systems, and plantsystems, including transgenic and transient plant systems.

In some embodiments, influenza antigens can be produced in plantsystems. Plants are relatively easy to manipulate genetically, and haveseveral advantages over alternative sources such as human fluids, animalcell lines, recombinant microorganisms and transgenic animals. Plantshave sophisticated post-translational modification machinery forproteins that is similar to that of mammals (although it should be notedthat there are some differences in glycosylation patterns between plantsand mammals). This enables production of bioactive reagents in planttissues. Also, plants can economically produce very large amounts ofbiomass without requiring sophisticated facilities. Moreover, plants arenot subject to contamination with animal pathogens. Like liposomes andmicrocapsules, plant cells are expected to provide protection forpassage of antigen to the gastrointestinal tract.

Plants can be utilized for production of heterologous proteins via useof various production systems. One such system includes use oftransgenic/genetically-modified plants where a gene encoding targetproduct is permanently incorporated into the genome of the plant.Transgenic systems can generate crop production systems. A variety offoreign proteins, including many of mammalian origin and many vaccinecandidate antigens, have been expressed in transgenic plants and shownto have functional activity (Tacket et al. (2000) J. Infect. Dis.182:302; and Thanavala et al. (2005) Proc. Natl. Acad. Sci. USA102:3378). Additionally, administration of unprocessed transgenic plantsexpressing hepatitis B major surface antigen to non-immunized humanvolunteers resulted in production of immune response (Kapusta et al.(1999) FASEB J. 13:1796).

Another system for expressing polypeptides in plants utilizes plantviral vectors engineered to express foreign sequences (e.g., transientexpression). This approach can allow for use of healthy non-transgenicplants as rapid production systems. Thus, genetically engineered plantsand plants infected with recombinant plant viruses can serve as “greenfactories” to rapidly generate and produce specific proteins ofinterest. Plant viruses have certain advantages that make themattractive as expression vectors for foreign protein production. Severalmembers of plant RNA viruses have been well characterized, andinfectious cDNA clones are available to facilitate genetic manipulation.Once infectious viral genetic material enters a susceptible host cell,it replicates to high levels and spreads rapidly throughout the entireplant. There are several approaches to producing target polypeptidesusing plant viral expression vectors, including incorporation of targetpolypeptides into viral genomes. One approach involves engineering coatproteins of viruses that infect bacteria, animals or plants to functionas carrier molecules for antigenic peptides. Such carrier proteins havethe potential to assemble and form recombinant virus-like particlesdisplaying desired antigenic epitopes on their surface. This approachallows for time-efficient production of antigen and/or antibodycandidates, since the particulate nature of an antigen and/or antibodycandidate facilitates easy and cost-effective recovery from planttissue. Additional advantages include enhanced target-specificimmunogenicity, the potential to incorporate multiple antigendeterminants and/or antibody sequences, and ease of formulation intoantigen and/or antibody that can be delivered nasally or parenterally,for example. As an example, spinach leaves containing recombinant plantviral particles carrying epitopes of virus fused to coat protein havegenerated immune response upon administration (Modelska et al. (1998)Proc. Natl. Acad. Sci. USA 95:2481; and Yusibov et al. (2002) Vaccine19/20:3155).

Plant Expression Systems

Any plant susceptible to incorporation and/or maintenance ofheterologous nucleic acid and capable of producing heterologous proteincan be utilized. In general, it will often be desirable to utilizeplants that are amenable to growth under defined conditions, for examplein a greenhouse and/or in aqueous systems. It may be desirable to selectplants that are not typically consumed by human beings or domesticatedanimals and/or are not typically part of the human food chain, so thatthey can be grown outside without concern that expressed polynucleotidemay be undesirably ingested. In some embodiments, however, it will bedesirable to employ edible plants. In particular embodiments, it will bedesirable to utilize plants that accumulate expressed polypeptides inedible portions of the plant.

Often, certain desirable plant characteristics will be determined by theparticular polynucleotide to be expressed. To give but a few examples,when a polynucleotide encodes a protein to be produced in high yield (aswill often be the case, for example, when antigen proteins are to beexpressed), it will often be desirable to select plants with relativelyhigh biomass (e.g., tobacco, which has additional advantages that it ishighly susceptible to viral infection, has a short growth period, and isnot in the human food chain). Where a polynucleotide encodes antigenprotein whose full activity requires (or is inhibited by) a particularpost-translational modification, the ability (or inability) of certainplant species to accomplish relevant modification (e.g., a particularglycosylation) may direct selection. For example, plants are capable ofaccomplishing certain post-translational modifications (e.g.,glycosylation), but plants will not generate sialation patterns whichare found in mammalian post-translational modification. Thus, plantproduction of antigen can result in production of a different entitythan the identical protein sequence produced in alternative systems.

In some embodiments, crop plants, or crop-related plants can be used. Insome cases, edible plants can be utilized.

Suitable plants include, without limitation, Angiosperms, Bryophytes(e.g., Hepaticae, Musci, etc.), Pteridophytes (e.g., ferns, horsetails,lycopods), Gymnosperms (e.g., conifers, cycase, Ginko, Gnetales), andAlgae (e.g., Chlorophyceae, Phaeophyceae, Rhodophyceae, Myxophyceae,Xanthophyceae, and Euglenophyceae). Exemplary plants are members of thefamily Leguminosae (Fabaceae; e.g., pea, alfalfa, soybean); Gramineae(Poaceae; e.g., corn, wheat, rice); Solanaceae, particularly of thegenus Lycopersicon (e.g., tomato), Solanum (e.g., potato, eggplant),Capsium (e.g., pepper), or Nicotiana (e.g., tobacco); Umbelliferae,particularly of the genus Daucus (e.g., carrot), Apium (e.g., celery),or Rutaceae (e.g., oranges); Compositae, particularly of the genusLactuca (e.g., lettuce); Brassicaceae (Cruciferae), particularly of thegenus Brassica or Sinapis. In certain aspects, exemplary plants can beplants of the Brassica or Arabidopsis genus. Some exemplary Brassicaceaefamily members include Brassica campestris, B. carinata, B. juncea, B.napus, B. nigra, B. oleraceae, B. tournifortii, Sinapis alba, andRaphanus sativus. Some suitable plants that are amendable totransformation and are edible as sprouted seedlings include alfalfa,mung bean, radish, wheat, mustard, spinach, carrot, beet, onion, garlic,celery, rhubarb, a leafy plant such as cabbage or lettuce, watercress orcress, herbs such as parsley, mint, or clovers, cauliflower, broccoli,soybean, lentils, edible flowers such as sunflower etc.

Introducing Vectors into Plants:

In general, vectors can be delivered to plants according to knowntechniques. For example, vectors themselves can be directly applied toplants (e.g., via abrasive inoculations, mechanized spray inoculations,vacuum infiltration, particle bombardment, or electroporation).Alternatively or additionally, virions can be prepared (e.g., fromalready infected plants), and can be applied to other plants accordingto known techniques.

A wide variety of viruses are known that infect various plant species,and can be employed for polynucleotide expression (see, for example, TheClassification and Nomenclature of Viruses, “Sixth Report of theInternational Committee on Taxonomy of Viruses,” Ed. Murphy et al.,Springer Verlag: New York, 1995, the entire contents of which areincorporated herein by reference; Grierson et al. Plant MolecularBiology, Blackie, London, pp. 126-146, 1984; Gluzman et al.,Communications in Molecular Biology: Viral Vectors, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., pp. 172-189, 1988; and Mathew,Plant Viruses Online, available online on the University of Idaho website at “image ‘dot’ fs ‘dot’ uidaho ‘dot’ edu ‘slash’ vide ‘slash’”).In some embodiments, rather than delivering a single viral vector to aplant cell, multiple different vectors can be delivered that, together,allow for replication (and, optionally cell-to-cell and/or long distancemovement) of viral vector(s). Some or all of the proteins can be encodedby the genome of transgenic plants. In certain aspects, described infurther detail herein, these systems include one or more viral vectorcomponents.

Vector systems that include components of two heterologous plant virusesin order to achieve a system that readily infects a wide range of planttypes and yet poses little or no risk of infectious spread. An exemplarysystem has been described previously (see, e.g., PCT Publication WO00/25574 and U.S. Patent Publication No. 2005/0026291, which areincorporated herein by reference). As noted herein, viral vectors can beapplied to plants (e.g., whole plants, portions of plants, sprouts,etc.) using various methods (e.g., through infiltration or mechanicalinoculation, spray, etc.). Where infection is to be accomplished bydirect application of a viral genome to a plant, any available techniquecan be used to prepare the genome. For example, many viruses that can beused have ssRNA genomes. ssRNA can be prepared by transcription of a DNAcopy of the genome, or by replication of an RNA copy, either in vivo orin vitro. Given the readily availability of easy-to-use in vitrotranscription systems (e.g., SP6, T7, reticulocyte lysate, etc.), andalso the convenience of maintaining a DNA copy of an RNA vector, it isexpected that ssRNA vectors often will be prepared by in vitrotranscription, particularly with T7 or SP6 polymerase.

In some embodiments, rather than introducing a single viral vector typeinto a plant, multiple different viral vectors can be introduced. Suchvectors can, for example, trans-complement each other with respect tofunctions such as replication, cell-to-cell movement, and/or longdistance movement. Vectors can contain different polynucleotidesencoding influenza antigens. Selection for plant(s) or portions thereofthat express multiple polypeptides encoding one or more influenzaantigen(s) can be performed as described above for singlepolynucleotides or polypeptides.

Plant Tissue Expression Systems:

As discussed above, influenza antigens can be produced in any suitablesystem. Vector constructs and expression systems are well known in theart and can be adapted to incorporate use of influenza antigens providedherein. For example, transgenic plant production is known and generationof constructs and plant production can be adapted according to knowntechniques in the art. In some embodiments, transient expression systemsin plants are desired. Two of these systems include production of clonalroots and clonal plant systems, and derivatives thereof, as well asproduction of sprouted seedlings systems.

Sprouts and Sprouted Seedling Plant Expression Systems:

Systems and reagents for generating a variety of sprouts and sproutedseedlings which are useful for production of influenza antigen(s) havebeen described previously and are known in the art (see, for example,PCT Publication WO 04/43886, which is incorporated herein by reference).This document further provides sprouted seedlings, which can be edible,as a biomass containing an influenza antigen. In certain aspects,biomass is provided directly for consumption of antigen containingcompositions. In some aspects, biomass is processed prior toconsumption, for example, by homogenizing, crushing, drying, orextracting. In certain aspects, influenza antigen is purified frombiomass and formulated into a pharmaceutical composition.

Additionally provided are methods for producing influenza antigen(s) insprouted seedlings that can be consumed or harvested live (e.g.,sprouts, sprouted seedlings of the Brassica genus). In some embodiments,the method can involve growing a seed to an edible sprouted seedling ina contained, regulatable environment (e.g., indoors, in a container,etc.). A seed can be a genetically engineered seed that contains anexpression cassette encoding an influenza antigen, which expression isdriven by an exogenously inducible promoter. A variety of promoters canbe used that are exogenously inducible by, for example, light, heat,phytohormones, or nutrients.

In some embodiments, methods of producing influenza antigen(s) insprouted seedlings can include first generating a seed stock for asprouted seedling by transforming plants with an expression cassettethat encodes influenza antigen using an Agrobacterium transformationsystem, wherein expression of an influenza antigen is driven by aninducible promoter. Transgenic seeds can be obtained from a transformedplant, grown in a contained, regulatable environment, and induced toexpress an influenza antigen.

In some embodiments, methods are provided that involves infectingsprouted seedlings with a viral expression cassette encoding aninfluenza antigen, expression of which can be driven by any of a viralpromoter or an inducible promoter. Sprouted seedlings can be grown fortwo to fourteen days in a contained, regulatable environment or at leastuntil sufficient levels of influenza antigen have been obtained forconsumption or harvesting.

This document further provides systems for producing influenzaantigen(s) in sprouted seedlings that include a housing unit withclimate control and a sprouted seedling containing an expressioncassette that encodes one or more influenza antigens, wherein expressionis driven by a constitutive or inducible promoter. The systems canprovide unique advantages over the outdoor environment or greenhouse,which cannot be controlled. Thus, a grower can precisely time theinduction of expression of influenza antigen, which can greatly reducetime and cost of producing influenza antigen(s).

In certain aspects, transiently transfected sprouts contain viral vectorsequences encoding an influenza antigen. Seedlings can be grown for atime period so as to allow for production of viral nucleic acid insprouts, followed by a period of growth wherein multiple copies of virusare produced, thereby resulting in production of influenza antigen(s).

In certain aspects, genetically engineered seeds or embryos that containa nucleic acid encoding influenza antigen(s) can be grown to sproutedseedling stage in a contained, regulatable environment. The contained,regulatable environment can be a housing unit or room in which seeds canbe grown indoors. All environmental factors of a contained, regulatableenvironment can be controlled. Since sprouts do not require light togrow, and lighting can be expensive, genetically engineered seeds orembryos can be grown to sprouted seedling stage indoors in the absenceof light.

Other environmental factors that can be regulated in a contained,regulatable environment include temperature, humidity, water, nutrients,gas (e.g., O₂ or CO₂ content or air circulation), chemicals (smallmolecules such as sugars and sugar derivatives or hormones such as suchas phytohormones gibberellic or absisic acid, etc.) and the like.

According to certain embodiments, expression of a nucleic acid encodingan influenza antigen can be controlled by an exogenously induciblepromoter. Exogenously inducible promoters can be caused to increase ordecrease expression of a nucleic acid in response to an external, ratherthan an internal stimulus. A number of environmental factors can act asinducers for expression of nucleic acids carried by expression cassettesof genetically engineered sprouts. A promoter can be a heat-induciblepromoter, such as a heat-shock promoter. For example, using asheat-shock promoter, temperature of a contained environment can simplybe raised to induce expression of a nucleic acid. Other promotersinclude light inducible promoters. Light-inducible promoters can bemaintained as constitutive promoters if light in a contained regulatableenvironment is always on. Alternatively or additionally, expression of anucleic acid can be turned on at a particular time during development bysimply turning on the light. A promoter can be a chemically induciblepromoter is used to induce expression of a nucleic acid. According tothese embodiments, a chemical could simply be misted or sprayed ontoseed, embryo, or seedling to induce expression of nucleic acid. Sprayingand misting can be precisely controlled and directed onto target seed,embryo, or seedling to which it is intended. The contained environmentis devoid of wind or air currents, which could disperse chemical awayfrom intended target, so that the chemical stays on the target for whichit was intended.

Time of expression can be induced can be selected to maximize expressionof an influenza antigen in sprouted seedling by the time of harvest.Inducing expression in an embryo at a particular stage of growth, forexample, inducing expression in an embryo at a particular number of daysafter germination, can result in maximum synthesis of an influenzaantigen at the time of harvest. For example, inducing expression fromthe promoter 4 days after germination can result in more proteinsynthesis than inducing expression from the promoter after 3 days orafter 5 days. Those skilled in the art will appreciate that maximizingexpression can be achieved by routine experimentation. In someembodiments, sprouted seedlings can be harvested at about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, or morethan 28 days after germination. In some embodiments, sprouted seedlingscan be harvested at about

In cases where the expression vector has a constitutive promoter insteadof an inducible promoter, sprouted seedling may be harvested at acertain time after transformation of sprouted seedling. For example, ifa sprouted seedling were virally transformed at an early stage ofdevelopment, for example, at embryo stage, sprouted seedlings may beharvested at a time when expression is at its maximumpost-transformation, e.g., at up to about 1 day, up to about 2 days, upto about 3 days, up to about 4 days, up to about 5 days, up to about 6days, up to about 7 days, up to about 8 days, up to about 9 days, up toabout 10 days, up to about 11 days, up to about 12 days, up to about 13days, up to about 14 days, up to about 15 days, up to about 16 days, upto about 17 days, up to about 18 days, up to about 19 days, up to about20 days, up to about 21 days, up to about 22 days, up to about 23 days,up to about 24 days, up to about 25 days, up to about 26 days, up toabout 27 days, up to about 28 days, up to about 29 days, up to about 30days post-transformation. It could be that sprouts develop one, two,three or more months post-transformation, depending on germination ofseed.

Generally, once expression of influenza antigen(s) begins, seeds,embryos, or sprouted seedlings can be allowed to grow until sufficientlevels of influenza antigen(s) are expressed. In certain aspects,sufficient levels can be levels that would provide a therapeutic benefitto a patient if harvested biomass were eaten raw. Alternatively oradditionally, sufficient levels can be levels from which influenzaantigen can be concentrated or purified from biomass and formulated intoa pharmaceutical composition that provides a therapeutic benefit to apatient upon administration. Typically, influenza antigen is not aprotein expressed in sprouted seedling in nature. At any rate, influenzaantigen is typically expressed at concentrations above that which wouldbe present in a sprouted seedling in nature.

Once expression of influenza antigen is induced, growth is allowed tocontinue until sprouted seedling stage, at which time sprouted seedlingscan be harvested. Sprouted seedlings can be harvested live. Harvestinglive sprouted seedlings has several advantages including minimal effortand breakage. Sprouted seedlings can be grown hydroponically, makingharvesting a simple matter of lifting the sprouted seedling from itshydroponic solution. No soil is required for growth of the sproutedseedlings, but may be provided if deemed necessary or desirable by theskilled artisan. Because sprouts can be grown without soil, no cleansingof sprouted seedling material is required at the time of harvest. Beingable to harvest the sprouted seedling directly from its hydroponicenvironment without washing or scrubbing minimizes breakage of theharvested material. Breakage and wilting of plants induces apoptosis.During apoptosis, certain proteolytic enzymes become active, which candegrade pharmaceutical protein expressed in the sprouted seedling,resulting in decreased therapeutic activity of the protein.Apoptosis-induced proteolysis can significantly decrease yield ofprotein from mature plants. Using methods as described herein, apoptosiscan be avoided when no harvesting takes place until the moment proteinsare extracted from the plant.

For example, live sprouts can be ground, crushed, or blended to producea slurry of sprouted seedling biomass, in a buffer containing proteaseinhibitors. Buffer can be maintained at about 4° C. In some aspects,sprouted seedling biomass is air-dried, spray dried, frozen, orfreeze-dried. As in mature plants, some of these methods, such asair-drying, can result in a loss of activity of pharmaceutical protein.However, because sprouted seedlings are very small and have a largesurface area to volume ratio, this is much less likely to occur. Thoseskilled in the art will appreciate that many techniques for harvestingbiomass that minimize proteolysis of expressed protein are available andcould be applied.

In some embodiments, sprouted seedlings can be edible. In certainembodiments, sprouted seedlings expressing sufficient levels ofinfluenza antigens can be consumed upon harvesting (e.g., immediatelyafter harvest, within minimal period following harvest) so thatabsolutely no processing occurs before sprouted seedlings are consumed.In this way, any harvest-induced proteolytic breakdown of influenzaantigen before administration of influenza antigen to a patient in needof treatment is minimized. For example, sprouted seedlings that areready to be consumed can be delivered directly to a patient.Alternatively or additionally, genetically engineered seeds or embryoscan be delivered to a patient in need of treatment and grown to sproutedseedling stage by a patient. In one aspect, a supply of geneticallyengineered sprouted seedlings is provided to a patient, or to a doctorwho will be treating patients, so that a continual stock of sproutedseedlings expressing certain desirable influenza antigens can becultivated. This can be particularly valuable for populations indeveloping countries, where expensive pharmaceuticals are not affordableor deliverable. The ease with which sprouted seedlings can be grownmakes sprouted seedlings particularly desirable for such developingpopulations.

The regulatable nature of the contained environment imparts advantagesover growing plants in the outdoor environment. In general, growinggenetically engineered sprouted seedlings that express pharmaceuticalproteins in plants provides a pharmaceutical product faster (becauseplants can be harvested younger) and with less effort, risk, andregulatory considerations than growing genetically engineered plants.The contained, regulatable environment can reduce or eliminate risk ofcross-pollinating plants in nature.

For example, a heat inducible promoter likely would not be used outdoorsbecause outdoor temperature cannot be controlled. The promoter would beturned on any time outdoor temperature rose above a certain level.Similarly, the promoter would be turned off every time outdoortemperature dropped. Such temperature shifts could occur in a singleday, for example, turning expression on in the daytime and off at night.A heat inducible promoter, such as those described herein, would noteven be practical for use in a greenhouse, which is susceptible toclimatic shifts to almost the same degree as outdoors. Growth ofgenetically engineered plants in a greenhouse is quite costly. Incontrast, in the present system, every variable can be controlled sothat the maximum amount of expression can be achieved with everyharvest.

In certain embodiments, sprouted seedlings can be grown in trays thatcan be watered, sprayed, or misted at any time during development ofsprouted seedling. For example, a tray can be fitted with one or morewatering, spraying, misting, and draining apparatus that can deliverand/or remove water, nutrients, chemicals etc. at specific time and atprecise quantities during development of a sprouted seedling. Forexample, seeds require sufficient moisture to keep them damp. Excessmoisture drains through holes in trays into drains in the floor of theroom. Typically, drainage water is treated as appropriate for removal ofharmful chemicals before discharge back into the environment.

Another advantage of trays is that they can be contained within a verysmall space. Since no light is required for sprouted seedlings to grow,trays containing seeds, embryos, or sprouted seedlings can be tightlystacked vertically on top of one another, providing a large quantity ofbiomass per unit floor space in a housing facility constructedspecifically for these purposes. In addition, stacks of trays can bearranged in horizontal rows within the housing unit. Once seedlings havegrown to a stage appropriate for harvest (about two to fourteen days)individual seedling trays can be moved into a processing facility,either manually or by automatic means, such as a conveyor belt.

The system is unique in that it provides a sprouted seedling biomass,which is a source of an influenza antigen(s). Whether consumed directlyor processed into the form of a pharmaceutical composition, becausesprouted seedlings can be grown in a contained, regulatable environment,sprouted seedling biomass and/or pharmaceutical composition derived frombiomass can be provided to a consumer at low cost. In addition, the factthat the conditions for growth of the sprouted seedlings can becontrolled makes the quality and purity of product consistent. Thecontained, regulatable environment can obviate many safety regulationsof the EPA that can prevent scientists from growing geneticallyengineered agricultural products outdoors.

Transformed Sprouts:

A variety of methods can be used to transform plant cells and producegenetically engineered sprouted seedlings. Two available methods fortransformation of plants that require that transgenic plant cell linesbe generated in vitro, followed by regeneration of cell lines into wholeplants include Agrobacterium tumefaciens mediated gene transfer andmicroprojectile bombardment or electroporation. Viral transformation isa more rapid and less costly method of transforming embryos and sproutedseedlings that can be harvested without an experimental or generationallag prior to obtaining desired product. For any of these techniques, theskilled artisan would appreciate how to adjust and optimizetransformation protocols that have traditionally been used for plants,seeds, embryos, or spouted seedlings.

Agrobacterium Transformation Expression Cassettes:

Agrobacterium is a representative genus of the gram-negative familyRhizobiaceae. This species is responsible for plant tumors such as crowngall and hairy root disease. In dedifferentiated plant tissue, which ischaracteristic of tumors, amino acid derivatives known as opines can beproduced by the Agrobacterium and catabolized by the plant. Thebacterial genes responsible for expression of opines are a convenientsource of control elements for chimeric expression cassettes. In somecases, an Agrobacterium transformation system can be used to generateedible sprouted seedlings, which can be merely harvested earlier thanmature plants. Agrobacterium transformation methods can easily beapplied to regenerate sprouted seedlings expressing influenza antigens.

In general, transforming plants involves transformation of plant cellsgrown in tissue culture by co-cultivation with an Agrobacteriumtumefaciens carrying a plant/bacterial vector. The vector contains agene encoding an influenza antigen. The Agrobacterium transfers vectorto plant host cell and is then eliminated using antibiotic treatment.Transformed plant cells expressing influenza antigen can be selected,differentiated, and finally regenerated into complete plantlets (Hellenset al. (2000) Plant Mol. Biol. 42:819; Pilon-Smits et al. (1999) PlantPhysiolog. 119:123; Barfield et al. (1991) Plant Cell Reports 10:308;and Riva et al. (1998) J. Biotech. 1(3).

Expression vectors can include a gene (or expression cassette) encodingan influenza antigen designed for operation in plants, with companionsequences upstream and downstream of the expression cassette. Thecompanion sequences generally can be of plasmid or viral origin andprovide necessary characteristics to the vector to transfer DNA frombacteria to the desired plant host.

The basic bacterial/plant vector construct can provide a broad hostrange prokaryote replication origin, a prokaryote selectable marker.Suitable prokaryotic selectable markers include resistance towardantibiotics such as ampicillin or tetracycline. Other DNA sequencesencoding additional functions that are well known in the art can bepresent in the vector.

Agrobacterium T-DNA sequences are required for Agrobacterium mediatedtransfer of DNA to the plant chromosome. The tumor-inducing genes ofT-DNA typically are removed and replaced with sequences encoding aninfluenza antigen. T-DNA border sequences can be retained because theyinitiate integration of the T-DNA region into the plant genome. Ifexpression of influenza antigen is not readily amenable to detection,the bacterial/plant vector construct can include a selectable markergene suitable for determining if a plant cell has been transformed,e.g., a nptII kanamycin resistance gene. On the same or differentbacterial/plant vector (Ti plasmid) are Ti sequences. Ti sequencesinclude virulence genes, which encode a set of proteins responsible forexcision, transfer and integration of T-DNA into the plant genome(Schell (1987) Science 237:1176). Other sequences suitable forpermitting integration of heterologous sequence into the plant genomecan include transposon sequences, and the like, for homologousrecombination.

Certain constructs will include an expression cassette encoding anantigen protein. One, two, or more expression cassettes can be used in agiven transformation. The recombinant expression cassette contains, inaddition to an influenza antigen encoding sequence, at least thefollowing elements: a promoter region, plant 5′ untranslated sequences,initiation codon (depending upon whether or not an expressed gene hasits own), and transcription and translation termination sequences. Inaddition, transcription and translation terminators can be included inexpression cassettes or chimeric genes. Signal secretion sequences thatallow processing and translocation of a protein, as appropriate, can beincluded in the expression cassette. A variety of promoters, signalsequences, and transcription and translation terminators are described(see, for example, Lawton et al. (1987) Plant Mol. Biol. 9:315; U.S.Pat. No. 5,888,789, incorporated herein by reference). In addition,structural genes for antibiotic resistance are commonly utilized as aselection factor (Fraley et al. (1983) Proc. Natl. Acad. Sci. USA80:4803, incorporated herein by reference). Unique restriction enzymesites at the 5′ and 3′ ends of a cassette allow for easy insertion intoa pre-existing vector. Other binary vector systems forAgrobacterium-mediated transformation, carrying at least one T-DNAborder sequence are described in PCT/EP99/07414, incorporated herein byreference.

Regeneration:

Seeds of transformed plants can be harvested, dried, cleaned, and testedfor viability and for the presence and expression of a desired geneproduct. Once this has been determined, seed stock is typically storedunder appropriate conditions of temperature, humidity, sanitation, andsecurity to be used when necessary. Whole plants then can be regeneratedfrom cultured protoplasts as described (see, e.g., Evans et al. Handbookof Plant Cell Cultures, Vol. 1: MacMillan Publishing Co. New York, 1983;and Vasil (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad.Press, Orlando, Fla., Vol. I, 1984, and Vol. III, 1986, incorporatedherein by reference). In certain aspects, plants can be regenerated onlyto sprouted seedling stage. In some aspects, whole plants can beregenerated to produce seed stocks and sprouted seedlings can begenerated from seeds of the seed stock.

All plants from which protoplasts can be isolated and cultured to givewhole, regenerated plants can be transformed so that whole plants can berecovered that contain a transferred gene. It is known that practicallyall plants can be regenerated from cultured cells or tissues, including,but not limited to, all major species of plants that produce ediblesprouts. Some suitable plants include Nicotiana species such as tobacco,alfalfa, mung bean, radish, wheat, mustard, spinach, carrot, beet,onion, garlic, celery, rhubarb, a leafy plant such as cabbage orlettuce, watercress or cress, herbs such as parsley, mint, or clovers,cauliflower, broccoli, soybean, lentils, and edible flowers such assunflower, etc.

Means for regeneration vary from one species of plants to the next.However, those skilled in the art will appreciate that generally asuspension of transformed protoplants containing copies of aheterologous gene is first provided. Callus tissue is formed and shootscan be induced from callus and subsequently rooted. Alternatively oradditionally, embryo formation can be induced from a protoplastsuspension. These embryos germinate as natural embryos to form plants.Steeping seed in water or spraying seed with water to increase themoisture content of the seed to between 35-45% initiates germination.For germination to proceed, seeds typically can be maintained in airsaturated with water under controlled temperature and airflowconditions. The culture media will generally contain various amino acidsand hormones, such as auxin and cytokinins. It is advantageous to addglutamic acid and proline to the medium, especially for such species asalfalfa. Shoots and roots normally develop simultaneously. Efficientregeneration will depend on the medium, the genotype, and the history ofthe culture. If these three variables can be controlled, thenregeneration is fully reproducible and repeatable.

The mature plants, grown from transformed plant cells, can be selfed andnon-segregating, homozygous transgenic plants can be identified. Aninbred plant can produce seeds containing antigen-encoding sequences.Such seeds can be germinated and grown to sprouted seedling stage toproduce influenza antigen(s).

In related embodiments, seeds can be formed into seed products and soldwith instructions on how to grow seedlings to the appropriate sproutedseedling stage for administration or harvesting into a pharmaceuticalcomposition. In some embodiments, hybrids or novel varieties embodyingdesired traits can be developed from inbred plants.

Direct Integration:

Direct integration of DNA fragments into the genome of plant cells bymicroprojectile bombardment or electroporation can be used (see, e.g.,Kikkert et al. (1999) In Vitro Cellular & Developmental Biology. Plant:Journal of the Tissue Culture Association 35:43; and Bates (1994) Mol.Biotech. 2:135). More particularly, vectors that express influenzaantigen(s) can be introduced into plant cells by a variety oftechniques. As described above, vectors can include selectable markersfor use in plant cells. Vectors can include sequences that allow theirselection and propagation in a secondary host, such as sequencescontaining an origin of replication and selectable marker. Typically,secondary hosts include bacteria and yeast. In one embodiment, asecondary host is bacteria (e.g., Escherichia coli, the origin ofreplication is a colE1-type origin of replication) and a selectablemarker is a gene encoding ampicillin resistance. Such sequences are wellknown in the art and are commercially available (e.g., Clontech, PaloAlto, Calif. or Stratagene, La Jolla, Calif.).

Vectors can be modified to intermediate plant transformation plasmidsthat contain a region of homology to an Agrobacterium tumefaciensvector, a T-DNA border region from Agrobacterium tumefaciens, andantigen encoding nucleic acids or expression cassettes described above.Further vectors can include a disarmed plant tumor inducing plasmid ofAgrobacterium tumefaciens.

Direct transformation of vectors can involve microinjecting vectorsdirectly into plant cells by use of micropipettes to mechanicallytransfer recombinant DNA (see, e.g., Crossway (1985) Mol. Gen. Genet.202:179). Genetic material can be transferred into a plant cell usingpolyethylene glycols (see, e.g., Krens et al. (1982) Nature 296:72).Another method of introducing nucleic acids into plants via highvelocity ballistic penetration by small particles with a nucleic acideither within the matrix of small beads or particles, or on the surface(see, e.g., Klein et al. (1987) Nature 327:70; and Knudsen et al. (1991)Planta 185:330). Yet another method of introduction is fusion ofprotoplasts with other entities, either minicells, cells, lysosomes, orother fusible lipid-surfaced bodies (see, e.g., Fraley et al. (1982)Proc. Natl. Acad. Sci. USA 79:1859). Vectors can be introduced intoplant cells by, for example, electroporation (see, e.g., Fromm et al.(1985) Proc. Natl. Acad. Sci. USA 82:5824). According to this technique,plant protoplasts can be electroporate in the presence of plasmidscontaining a gene construct. Electrical impulses of high field strengthreversibly permeabilize biomembranes allowing introduction of plasmids.Electroporated plant protoplasts reform the cell wall divide and formplant callus, which can be regenerated to form sprouted seedlings. Thoseskilled in the art will appreciate how to utilize these methods totransform plants cells that can be used to generate edible sproutedseedlings.

Viral Transformation:

Similar to conventional expression systems, plant viral vectors can beused to produce full-length proteins, including full length antigen.Plant virus vectors can be used to infect and produce antigen(s) inseeds, embryos, sprouted seedlings, etc. Viral system that can be usedto express everything from short peptides to large complex proteins.Specifically, using tobamoviral vectors is described (see, for example,McCormick et al. (1999) Proc. Natl. Acad. Sci. USA 96:703; Kumagai etal. (2000) Gene 245:169; and Verch et al. (1998) J. Immunol. Methods220:69). Thus, plant viral vectors have a demonstrated ability toexpress short peptides as well as large complex proteins.

In certain embodiments, transgenic sprouts, which express influenzaantigen, can be generated utilizing a host/virus system. Transgenicsprouts produced by viral infection provide a source of transgenicprotein that has already been demonstrated to be safe. For example,sprouts are free of contamination with animal pathogens. In addition, avirus/sprout system offers a much simpler, less expensive route forscale-up and manufacturing, since transgenes can be introduced intovirus, which can be grown up to a commercial scale within a few days. Incontrast, transgenic plants can require up to 5-7 years beforesufficient seeds or plant material is available for large-scale trialsor commercialization.

Plant RNA viruses can have certain advantages that make them attractiveas vectors for foreign protein expression. The molecular biology andpathology of a number of plant RNA viruses are well characterized andthere is considerable knowledge of virus biology, genetics, andregulatory sequences. Most plant RNA viruses have small genomes andinfectious cDNA clones are available to facilitate genetic manipulation.Once infectious virus material enters a susceptible host cell, itreplicates to high levels and spreads rapidly throughout the entiresprouted seedling (one to ten days post inoculation). Virus particlesare easily and economically recovered from infected sprouted seedlingtissue. Viruses have a wide host range, enabling use of a singleconstruct for infection of several susceptible species. Thesecharacteristics are readily transferable to sprouts.

Foreign sequences can be expressed from plant RNA viruses, typically byreplacing one of viral genes with desired sequence, by inserting foreignsequences into the virus genome at an appropriate position, or by fusingforeign peptides to structural proteins of a virus. Moreover, any ofthese approaches can be combined to express foreign sequences bytrans-complementation of vital functions of a virus. A number ofdifferent strategies exist as tools to express foreign sequences invirus-infected plants using tobacco mosaic virus (TMV), alfalfa mosaicvirus (AlMV), and chimeras thereof.

TMV, the prototype of tobamoviruses, has a genome consisting of a singleplus-sense RNA encapsidated with a 17.0 kD CP, which results inrod-shaped particles (300 nm in length). CP is the only structuralprotein of TMV and is required for encapsidation and long distancemovement of virus in an infected host (Saito et al. (1990) Virology176:329). 183 and 126 kD proteins are translated from genomic RNA andare required for virus replication (Ishikawa et al. (1986) Nucleic AcidsRes. 14:8291). 30 kD protein is the cell-to-cell movement protein ofvirus (Meshi et al. (1987) EMBO J. 6:2557). Movement and coat proteinsare translated from subgenomic mRNAs (Hunter et al. (1976) Nature260:759; Bruening et al. (1976) Virology 71:498; and Beachy et al.(1976) Virology 73:498; each of which is incorporated herein byreference).

Other methods of transforming plant tissues include transforming theflower of the plant. Transformation of Arabidopsis thaliana can beachieved by dipping plant flowers into a solution of Agrobacteriumtumefaciens (Curtis et al. (2001) Transgenic Research 10:363; Qing etal. (2000) Molecular Breeding: New Strategies in Plant Improvement1:67). Transformed plants can be formed in the population of seedsgenerated by “dipped” plants. At a specific point during flowerdevelopment, a pore exists in the ovary wall through which Agrobacteriumtumefaciens gains access to the interior of the ovary. Once inside theovary, the Agrobacterium tumefaciens proliferates and transformsindividual ovules (Desfeux et al. (2000) Plant Physiol. 123:895).Transformed ovules follow the typical pathway of seed formation withinthe ovary.

Production and Isolation of Antigen:

In general, standard methods known in the art can be used for culturingor growing plants, plant cells, and/or plant tissues (e.g., clonalplants, clonal plant cells, leaves, sprouts, and sprouted seedlings) forproduction of antigen(s). A wide variety of culture media andbioreactors have been employed to culture hairy root cells, root celllines, and plant cells (see, for example, Giri et al. (2000) Biotechnol.Adv. 18:1; Rao et al. (2002) Biotechnol. Adv. 20:101; and references inboth of the foregoing, all of which are incorporated herein byreference. Clonal plants can be grown in any suitable manner.

In some embodiments, an influenza antigen can be expressed in a plant orportion thereof. Proteins can be isolated and purified in accordancewith conventional conditions and techniques known in the art. Theseinclude methods such as extraction, precipitation, chromatography,affinity chromatography, electrophoresis, and the like. This documentprovides for purification and affordable scaling up of production ofinfluenza antigen(s) using any of a variety of plant expression systemsknown in the art and provided herein, including viral plant expressionsystems described herein.

In some embodiments, it can be desirable to isolate influenza antigen(s)for generation of antibody products and/or desirable to isolateinfluenza antibody or antigen binding fragment produced. Where a proteinis produced from plant tissue(s) or a portion thereof, e.g., roots, rootcells, plants, plant cells, that express them, methods described infurther detail herein, or any applicable methods known in the art can beused for any of partial or complete isolation from plant material. Whereit is desirable to isolate the expression product from some or all ofplant cells or tissues that express it, any available purificationtechniques can be employed. Those of ordinary skill in the art arefamiliar with a wide range of fractionation and separation procedures(see, for example, Scopes et al., Protein Purification: Principles andPractice, 3^(rd) Ed., Janson et al., 1993; Protein Purification:Principles, High Resolution Methods, and Applications, Wiley-VCH, 1998;Springer-Verlag, NY, 1993; and Roe, Protein Purification Techniques,Oxford University Press, 2001; each of which is incorporated herein byreference). In some embodiments, it can be desirable to render theproduct more than about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% pure. See, e.g., U.S. Pat. Nos.6,740,740 and 6,841,659 for discussion of certain methods useful forpurifying substances from plant tissues or fluids.

Those skilled in the art will appreciate that a method of obtainingdesired influenza antigen(s) product(s) is by extraction. Plant material(e.g., roots, leaves, etc.) can be extracted to remove desired productsfrom residual biomass, thereby increasing the concentration and purityof product. Plants can be extracted in a buffered solution. For example,plant material can be transferred into an amount of ice-cold water at aratio of one to one by weight that has been buffered with, e.g.,phosphate buffer. Protease inhibitors can be added as required. Plantmaterial can be disrupted by vigorous blending or grinding whilesuspended in buffer solution and extracted biomass removed by filtrationor centrifugation. The product carried in solution can be furtherpurified by additional steps or converted to a dry powder byfreeze-drying or precipitation. Extraction can be carried out bypressing. Plants or roots can be extracted by pressing in a press or bybeing crushed as they are passed through closely spaced rollers. Fluidsexpressed from crushed plants or roots are collected and processedaccording to methods well known in the art. Extraction by pressingallows release of products in a more concentrated form. However, overallyield of product may be lower than if product were extracted insolution.

Antibodies

This document provides anti-influenza neuraminidase antibodies that canbe used, for example, for therapeutic and/or prophylactic purposes, suchas treatment of influenza infection. In some embodiments, anti-influenzaantibodies can be produced by plant(s) or portions thereof (e.g., roots,cells, sprouts, or cell line), using materials and methods describedherein, for example. In some cases, influenza antibodies can beexpressed in plants, plant cells, and/or plant tissues (e.g., sprouts,sprouted seedlings, leaves, roots, root culture, clonal cells, clonalcell lines, and clonal plants), and can be used directly from plant orpartially purified or purified in preparation for pharmaceuticaladministration to a subject.

Monoclonal Antibodies:

Various methods for generating monoclonal antibodies (mAbs) are wellknown in the art. See, e.g., the methods described in U.S. Pat. No.4,196,265, incorporated herein by reference. The most standardmonoclonal antibody generation techniques generally begin along the samelines as those for preparing polyclonal antibodies (Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, 1988, which is herebyincorporated by reference). Typically, a suitable animal can beimmunized with a selected immunogen to stimulate antibody-producingcells. Rodents such as mice and rats are exemplary animals, althoughrabbits, sheep, frogs, and chickens also can be used. Mice can beparticularly useful (e.g., BALB/c mice are routinely used and generallygive a higher percentage of stable fusions).

Following immunization, somatic cells with the potential for producingthe desired antibodies, specifically B lymphocytes (B cells), can beselected for use in MAb generation and fusion with cells of an immortalmyeloma cell, generally one of the same species as the animal that wasimmunized. Myeloma cell lines suited for use in hybridoma-producingfusion procedures typically are non-antibody-producing, have high fusionefficiency, and enzyme deficiencies that render then incapable ofgrowing in certain selective media which support the growth of only thedesired fused cells (hybridomas). Any one of a number of myeloma cellscan be used, as are known to those of skill in the art. For example,where the immunized animal is a mouse, one can use P3-X63/Ag8,X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG1.7 and S194/5XX0 Bul; for rats, one can use R210.RCY3, Y3-Ag 1.2.3,IR983F, 4B210 or one of the above listed mouse cell lines. U-266,GM1500-GRG2, LICR-LON-HMy2 and UC729-6, all can be useful in connectionwith human cell fusions.

This culturing can provide a population of hybridomas from whichspecific hybridomas can be selected, followed by serial dilution andcloning into individual antibody producing lines, which can bepropagated indefinitely for production of antibody.

MAbs produced generally can be further purified, e.g., using filtration,centrifugation and various chromatographic methods, such as HPLC oraffinity chromatography, all of which purification techniques are wellknown to those of skill in the art. These purification techniques eachinvolve fractionation to separate the desired antibody from othercomponents of a mixture. Analytical methods particularly suited to thepreparation of antibodies include, for example, protein A-Sepharoseand/or protein G-Sepharose chromatography.

As described in the Examples below, the 2B9 anti-N1NA monoclonalantibody has a light chain amino acid sequence as set forth in SEQ IDNO:5 and a heavy chain amino acid sequence as set forth in SEQ ID NO:6.The variable region of the light chain is encoded by amino acids 1-127of SEQ ID NO:5, and the variable region of the heavy chain is encoded byamino acids 1-137 of SEQ ID NO:6. Thus, this document provides anisolated monoclonal antibody that binds neuraminidase, wherein theantibody includes a light chain variable region amino acid sequence asset forth in amino acids 1 to 127 of SEQ ID NO:5, and a heavy chainvariable region amino acid sequence as set forth in amino acids 1 to 137of SEQ ID NO:6, and wherein the antibody has the ability to inhibitneuraminidase enzyme activity. The ability of a particular antibody toinhibit neuraminidase activity can be evaluated using, for example, themethods disclosed in the Examples herein.

Antibody Fragments and Derivatives:

Irrespective of the source of the original antibody against aneuraminidase, either the intact antibody, antibody multimers, or anyone of a variety of functional, antigen-binding regions of the antibodycan be used. Exemplary functional regions include scFv, Fv, Fab′, Faband F(ab′)₂ fragments of antibodies. Techniques for preparing suchconstructs are well known to those in the art and are furtherexemplified herein.

The choice of antibody construct can be influenced by various factors.For example, prolonged half-life can result from the active readsorptionof intact antibodies within the kidney, a property of the Fc piece ofimmunoglobulin. IgG based antibodies, therefore, are expected to exhibitslower blood clearance than their Fab′ counterparts. However, Fab′fragment-based compositions will generally exhibit better tissuepenetrating capability.

Antibody fragments can be obtained by proteolysis of the wholeimmunoglobulin by the non-specific thiolprotease, papain. Papaindigestion yields two identical antigen-binding fragments, termed “Fabfragments,” each with a single antigen-binding site, and a residual “Fcfragment.” The various fractions can be separated by protein A-Sepharoseor ion exchange chromatography.

The usual procedure for preparation of F(ab′)₂ fragments from IgG ofrabbit and human origin is limited proteolysis by the enzyme pepsin.Pepsin treatment of intact antibodies yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

A Fab fragment contains the constant domain of the light chain and thefirst constant domain (CH1) of the heavy chain. Fab′ fragments differfrom Fab fragments by the addition of a few residues at the carboxylterminus of the heavy chain CH1 domain including one or more cysteine(s)from the antibody hinge region. F(ab′)₂ antibody fragments wereoriginally produced as pairs of Fab′ fragments that have hinge cysteinesbetween them. Other chemical couplings of antibody fragments are known.

An “Fv” fragment is the minimum antibody fragment that contains acomplete antigen-recognition and binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,con-covalent association. It is in this configuration that threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimerCollectively, six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

“Single-chain Fv” or “scFv” antibody fragments (now known as “singlechains”) comprise the V_(H) and V_(L) domains of an antibody, whereinthese domains can be present in a single polypeptide chain. Generally,the Fv polypeptide further comprises a polypeptide linker between V_(H)and V_(L) domains that enables sFv to form the desired structure forantigen binding.

The following patents are incorporated herein by reference for thepurposes of even further supplementing the present teachings regardingthe preparation and use of functional, antigen-binding regions ofantibodies, including scFv, Fv, Fab′, Fab and F(ab′)₂ fragments ofantibodies: U.S. Pat. Nos. 5,855,866; 5,877,289; 5,965,132; 6,093,399;6,261,535; and 6,004,555. WO 98/45331 is also incorporated herein byreference for purposes including even further describing and teachingthe preparation of variable, hypervariable and complementaritydetermining (CDR) regions of antibodies.

“Diabodies” are small antibody fragments with two antigen-binding sites,which fragments comprise a heavy chain variable domain (V_(H)) connectedto a light chain variable domain (V_(L)) in the same polypeptide chain(V_(H)-V_(L)). By using a linker that is too short to allow pairingbetween two domains on the same chain, the domains can be forced to pairwith the complementary domains of another chain and create twoantigen-binding sites. Diabodies are described in EP 404,097 and WO93/11161, each of which is incorporated herein by reference. “Linearantibodies,” which can be bispecific or monospecific, comprise a pair oftandem Fd segments (V_(H)-C_(H)1-V_(H)-C_(H)1) that form a pair ofantigen binding regions, as described (see, for example, Zapata et al.(1995) Prot. Eng. 8:1057, incorporated herein by reference).

In using a Fab′ or antigen binding fragment of an antibody, with theattendant benefits on tissue penetration, one can derive additionaladvantages from modifying the fragment to increase its half-life. Avariety of techniques can be employed, such as manipulation ormodification of the antibody molecule itself, and conjugation to inertcarriers. Any conjugation for the sole purpose of increasing half-life,rather than to deliver an agent to a target, should be approachedcarefully in that Fab′ and other fragments can be chosen to penetratetissues. Nonetheless, conjugation to non-protein polymers, such PEG andthe like, is contemplated.

Modifications other than conjugation therefore are based upon modifyingthe structure of the antibody fragment to render it more stable, and/orto reduce the rate of catabolism in the body. One mechanism for suchmodifications is the use of D-amino acids in place of L-amino acids.Those of ordinary skill in the art will understand that the introductionof such modifications needs to be followed by rigorous testing of theresultant molecule to ensure that it still retains the desiredbiological properties. Further stabilizing modifications include the useof the addition of stabilizing moieties to either N-terminal orC-terminal, or both, which is generally used to prolong half-life ofbiological molecules. By way of example only, one may wish to modifytermini by acylation or amination.

Bispecific Antibodies:

Bispecific antibodies in general can be employed, so long as one armbinds to an aminophospholipid or anionic phospholipid and the bispecificantibody is attached, at a site distinct from the antigen binding site,to a therapeutic agent.

In general, the preparation of bispecific antibodies is well known inthe art. One method involves the separate preparation of antibodieshaving specificity for the aminophospholipid or anionic phospholipid, onthe one hand, and a therapeutic agent on the other. Peptic F(ab′)₂fragments can be prepared from two chosen antibodies, followed byreduction of each to provide separate Fab′_(SH) fragments. SH groups onone of two partners to be coupled then can be alkylated with across-linking reagent such as O-phenylenedimaleimide to provide freemaleimide groups on one partner. This partner then can be conjugated tothe other by means of a thioether linkage, to give the desired F(ab′)₂heteroconjugate. Other techniques are known wherein cross-linking withSPDP or protein A is carried out, or a trispecific construct isprepared.

One method for producing bispecific antibodies is by the fusion of twohybridomas to form a quadroma. As used herein, the term “quadroma” isused to describe the productive fusion of two B cell hybridomas. Usingnow standard techniques, two antibody producing hybridomas can be fusedto give daughter cells, and those cells that have maintained theexpression of both sets of clonotype immunoglobulin genes then can beselected.

CDR Technologies:

Antibodies are comprised of variable and constant regions. The term“variable,” as used herein in reference to antibodies, means thatcertain portions of the variable domains differ extensively in sequenceamong antibodies, and are used in the binding and specificity of eachparticular antibody to its particular antigen. However, the variabilityis concentrated in three segments termed “hypervariable regions,” bothin the light chain and the heavy chain variable domains.

The more highly conserved portions of variable domains are called theframework region (FR). Variable domains of native heavy and light chainseach comprise four FRs (FR1, FR2, FR3 and FR4, respectively), largelyadopting a beta-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases, forming partof, the beta-sheet structure.

The hypervariable regions in each chain are held together in closeproximity by the FRs and, with hypervariable regions from the otherchain, contribute to the formation of the antigen-binding site ofantibodies (Kabat et al. (1991), Sequences of proteins of immunologicalinterest, 5th ed. Bethesda, Md.: National Institutes of Health,incorporated herein by reference). Constant domains are not involveddirectly in binding an antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “hypervariable region,” as used herein, refers to amino acidresidues of an antibody that are responsible for antigen-binding. Thehypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-56 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al. (1991), supra) and/or those residues from a “hypervariableloop” (i.e. residues 26-32 (L1), 50-52(L2) and 91-96 (L3) in the lightchain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in theheavy chain variable domain). “Framework” or “FR” residues are thosevariable domain residues other than the hypervariable region residues asherein defined.

The DNA and deduced amino acid sequences of Vh and V kappa chains of the2B9 antibody encompass CDR1-3 of variable regions of heavy and lightchains of the antibody. In light of the sequence and other informationprovided herein, and the knowledge in the art, a range of 2B9-like andimproved antibodies and antigen binding regions can now be prepared andare thus encompassed herein.

In some embodiments, this document provides at least one CDR of theantibody produced by the hybridoma 2B9. In some embodiments, thisdocument provides a CDR, antibody, or antigen binding region thereof,which binds to at least a neuraminidase, and which comprises at leastone CDR of the antibody produced by the hybridoma 2B9.

In a particular embodiment, this document provides an antibody orantigen binding region thereof in which the framework regions of the 2B9antibody have been changed from mouse to a human IgG, such as human IgG1or other IgG subclass to reduce immunogenicity in humans. In someembodiments, sequences of the 2B9 antibody can be examined for thepresence of T-cell epitopes, as is known in the art. The underlyingsequence can then be changed to remove T-cell epitopes, i.e., to“deimmunize” the antibody.

The availability of DNA and amino acid sequences of Vh and V kappachains of the 2B9 antibody means that a range of antibodies can now beprepared using CDR technologies. In particular, random mutations can bemade in the CDRs and products screened to identify antibodies withhigher affinities and/or higher specificities. Such mutagenesis andselection is routinely practiced in the antibody arts. These methods canbe particularly suitable for use in the methods described herein, giventhe advantageous screening techniques disclosed herein. A convenient wayfor generating such substitutional variants is affinity maturation usingphage display.

CDR shuffling and implantation technologies can be used with the 2B9antibodies provided herein, for example. CDR shuffling inserts CDRsequences into a specific framework region (Jirholt et al. (1998) Gene215:471, incorporated herein by reference). CDR implantation techniquespermit random combination of CDR sequences into a single masterframework (Soderlind et al. (1999) Immunotechnol. 4:279; and Soderlindet al. (2000) Nature Biotechnol. 18:852, each incorporated herein byreference). Using such techniques, CDR sequences of the 2B9 antibody,for example, can be mutagenized to create a plurality of differentsequences, which can be incorporated into a scaffold sequence and theresultant antibody variants screened for desired characteristics, e.g.,higher affinity.

Antibodies from Phagemid Libraries:

Recombinant technology allows for preparation of antibodies having adesired specificity from recombinant genes encoding a range ofantibodies. Certain recombinant techniques involve isolation of antibodygenes by immunological screening of combinatorial immunoglobulin phageexpression libraries prepared from RNA isolated from spleen of animmunized animal (Morrison et al. (1986) Mt. Sinai J. Med. 53:175;Winter and Milstein (1991) Nature 349:293; Barbas et al. (1992) Proc.Natl. Acad. Sci. USA 89:4457; each incorporated herein by reference).For such methods, combinatorial immunoglobulin phagemid libraries can beprepared from RNA isolated from spleen of an immunized animal, andphagemids expressing appropriate antibodies can be selected by panningusing cells expressing antigen and control cells. Advantage of thisapproach over conventional hybridoma techniques include approximately10⁴ times as many antibodies can be produced and screened in a singleround, and that new specificities can be generated by H and L chaincombination, which can further increase the percentage of appropriateantibodies generated.

One method for the generation of a large repertoire of diverse antibodymolecules in bacteria utilizes the bacteriophage lambda as the vector(Huse et al. (1989) Science 246:1275; incorporated herein by reference).Production of antibodies using the lambda vector involves the cloning ofheavy and light chain populations of DNA sequences into separatestarting vectors. Vectors subsequently can be randomly combined to forma single vector that directs co-expression of heavy and light chains toform antibody fragments. The general technique for filamentous phagedisplay is described (U.S. Pat. No. 5,658,727, incorporated herein byreference). In a most general sense, the method provides a system forthe simultaneous cloning and screening of pre-selected ligand-bindingspecificities from antibody gene repertoires using a single vectorsystem. Screening of isolated members of the library for a pre-selectedligand-binding capacity allows the correlation of the binding capacityof an expressed antibody molecule with a convenient means to isolate agene that encodes the member from the library. Additional methods forscreening phagemid libraries are described (U.S. Pat. Nos. 5,580,717;5,427,908; 5,403,484; and 5,223,409, each incorporated herein byreference).

One method for the generation and screening of large libraries of whollyor partially synthetic antibody combining sites, or paratopes, utilizesdisplay vectors derived from filamentous phage such as M13, fl or fd(U.S. Pat. No. 5,698,426, incorporated herein by reference). Filamentousphage display vectors, referred to as “phagemids,” yield large librariesof monoclonal antibodies having diverse and novel immunospecificities.The technology uses a filamentous phage coat protein membrane anchordomain as a means for linking gene-product and gene during the assemblystage of filamentous phage replication, and has been used for thecloning and expression of antibodies from combinatorial libraries (Kanget al. (1991) Proc. Natl. Acad. Sci. USA 88:4363; and Barbas et al.(1991) Proc. Natl. Acad. Sci. USA 88:7978; each incorporated herein byreference). The surface expression library is screened for specific Fabfragments that bind neuraminidase molecules by standard affinityisolation procedures. The selected Fab fragments can be characterized bysequencing the nucleic acids encoding the polypeptides afteramplification of the phage population.

One method for producing diverse libraries of antibodies and screeningfor desirable binding specificities is described (U.S. Pat. Nos.5,667,988 and 5,759,817, each incorporated herein by reference). Themethod involves the preparation of libraries of heterodimericimmunoglobulin molecules in the form of phagemid libraries usingdegenerate oligonucleotides and primer extension reactions toincorporate degeneracies into CDR regions of immunoglobulin variableheavy and light chain variable domains, and display of mutagenizedpolypeptides on the surface of the phagemid. Thereafter, the displayprotein is screened for the ability to bind to a preselected antigen. Afurther variation of this method for producing diverse libraries ofantibodies and screening for desirable binding specificities isdescribed U.S. Pat. No. 5,702,892, incorporated herein by reference). Inthis method, only heavy chain sequences are employed, heavy chainsequences are randomized at all nucleotide positions that encode eitherthe CDR1 or CDRIII hypervariable region, and the genetic variability inthe CDRs can be generated independent of any biological process.

Transgenic Mice Containing Human Antibody Libraries:

Recombinant technology is available for the preparation of antibodies.In addition to the combinatorial immunoglobulin phage expressionlibraries disclosed above, one molecular cloning approach is to prepareantibodies from transgenic mice containing human antibody libraries.Such techniques are described (U.S. Pat. No. 5,545,807, incorporatedherein by reference).

In a most general sense, these methods involve the production of atransgenic animal that has inserted into its germline genetic materialthat encodes for at least part of an immunoglobulin of human origin orthat can rearrange to encode a repertoire of immunoglobulins. Theinserted genetic material can be produced from a human source, or can beproduced synthetically. The material can code for at least part of aknown immunoglobulin or can be modified to code for at least part of analtered immunoglobulin.

The inserted genetic material is expressed in the transgenic animal,resulting in production of an immunoglobulin derived at least in partfrom the inserted human immunoglobulin genetic material. The insertedgenetic material can be in the form of DNA cloned into prokaryoticvectors such as plasmids and/or cosmids. Larger DNA fragments can beinserted using yeast artificial chromosome vectors (Burke et al. (1987)Science 236:806; incorporated herein by reference), or by introductionof chromosome fragments. The inserted genetic material can be introducedto the host in conventional manner, for example by injection or otherprocedures into fertilized eggs or embryonic stem cells.

Once a suitable transgenic animal has been prepared, the animal issimply immunized with the desired immunogen. Depending on the nature ofthe inserted material, the animal can produce a chimeric immunoglobulin,e.g. of mixed mouse/human origin, where the genetic material of foreignorigin encodes only part of the immunoglobulin; or the animal canproduce an entirely foreign immunoglobulin, e.g. of wholly human origin,where the genetic material of foreign origin encodes an entireimmunoglobulin.

Polyclonal antisera can be produced from the transgenic animal followingimmunization. Immunoglobulin-producing cells can be removed from theanimal to produce the immunoglobulin of interest. Generally, monoclonalantibodies can be produced from the transgenic animal, e.g., by fusingspleen cells from the animal with myeloma cells and screening theresulting hybridomas to select those producing the desired antibody.Suitable techniques for such processes are described herein.

In one approach, the genetic material can be incorporated in the animalin such a way that the desired antibody is produced in body fluids suchas serum or external secretions of the animal, such as milk, colostrumor saliva. For example, by inserting in vitro genetic material encodingfor at least part of a human immunoglobulin into a gene of a mammalcoding for a milk protein and then introducing the gene to a fertilizedegg of the mammal, e.g., by injection, the egg can develop into an adultfemale mammal producing milk containing immunoglobulin derived at leastin part from the inserted human immunoglobulin genetic material. Thedesired antibody can then be harvested from the milk. Suitabletechniques for carrying out such processes are known to those skilled inthe art.

The foregoing transgenic animals can be employed to produce humanantibodies of a single isotype, more specifically an isotype that isessential for B cell maturation, such as IgM and possibly IgD. Anothermethod for producing human antibodies is described in U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,770,429;each incorporated by reference, wherein transgenic animals are describedthat are capable of switching from an isotype needed for B celldevelopment to other isotypes.

In the method described in U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,661,016; and 5,770,429, human immunoglobulintransgenes contained within a transgenic animal function correctlythroughout the pathway of B-cell development, leading to isotypeswitching. Accordingly, in this method, these transgenes are constructedso as to produce isotype switching and one or more of the following: (1)high level and cell-type specific expression, (2) functional generearrangement, (3) activation of and response to allelic exclusion, (4)expression of a sufficient primary repertoire, (5) signal transduction,(6) somatic hypermutation, and (7) domination of the transgene antibodylocus during the immune response.

Humanized Antibodies:

Human antibodies generally have at least three potential advantages foruse in human therapy. First, because the effector portion is human, itcan interact better with other parts of the human immune system, e.g.,to destroy target cells more efficiently by complement-dependentcytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC).Second, the human immune system should not recognize the antibody asforeign. Third, half-life in human circulation will be similar tonaturally occurring human antibodies, allowing smaller and less frequentdoses to be given.

Various methods for preparing human antibodies are provided herein. Inaddition to human antibodies, “humanized” antibodies have manyadvantages. “Humanized” antibodies are generally chimeric or mutantmonoclonal antibodies from mouse, rat, hamster, rabbit or other species,bearing human constant and/or variable region domains or specificchanges. Techniques for generating a so-called “humanized” antibody arewell known to those of skill in the art.

A number of methods have been described to produce humanized antibodies.Controlled rearrangement of antibody domains joined through proteindisulfide bonds to form new, artificial protein molecules or “chimeric”antibodies can be utilized (Konieczny et al. (1981) Haematologia(Budap.) 14:95; incorporated herein by reference). Recombinant DNAtechnology can be used to construct gene fusions between DNA sequencesencoding mouse antibody variable light and heavy chain domains and humanantibody light and heavy chain constant domains (Morrison et al. (1984)Proc. Natl. Acad. Sci. USA 81:6851; incorporated herein by reference).

DNA sequences encoding antigen binding portions or complementaritydetermining regions (CDR's) of murine monoclonal antibodies can begrafted by molecular means into DNA sequences encoding frameworks ofhuman antibody heavy and light chains (Jones et al. (1986) Nature321:522; Riechmann et al. (1988) Nature 332:323; each incorporatedherein by reference). Expressed recombinant products are called“reshaped” or humanized antibodies, and comprise the framework of ahuman antibody light or heavy chain and antigen recognition portions,CDR's, of a murine monoclonal antibody.

One method for producing humanized antibodies is described in U.S. Pat.No. 5,639,641, incorporated herein by reference. A similar method forthe production of humanized antibodies is described in U.S. Pat. Nos.5,693,762; 5,693,761; 5,585,089; and 5,530,101, each incorporated hereinby reference. These methods involve producing humanized immunoglobulinshaving one or more complementarity determining regions (CDR's) andpossible additional amino acids from a donor immunoglobulin and aframework region from an accepting human immunoglobulin. Each humanizedimmunoglobulin chain usually comprises, in addition to CDR's, aminoacids from the donor immunoglobulin framework that are capable ofinteracting with CDR's to effect binding affinity, such as one or moreamino acids that are immediately adjacent to a CDR in the donorimmunoglobulin or those within about 3 A as predicted by molecularmodeling. Heavy and light chains can each be designed by using any one,any combination, or all of various position criteria described in U.S.Pat. Nos. 5,693,762; 5,693,761; 5,585,089; and 5,530,101, eachincorporated herein by reference. When combined into an intact antibody,humanized immunoglobulins can be substantially non-immunogenic in humansand retain substantially the same affinity as the donor immunoglobulinto the original antigen.

An additional method for producing humanized antibodies is described inU.S. Pat. Nos. 5,565,332 and 5,733,743, each incorporated herein byreference. This method combines the concept of humanizing antibodieswith the phagemid libraries described herein. In a general sense, themethod utilizes sequences from the antigen binding site of an antibodyor population of antibodies directed against an antigen of interest.Thus for a single rodent antibody, sequences comprising part of theantigen binding site of the antibody can be combined with diverserepertoires of sequences of human antibodies that can, in combination,create a complete antigen binding site.

Antigen binding sites created by this process differ from those createdby CDR grafting, in that only the portion of sequence of the originalrodent antibody is likely to make contacts with antigen in a similarmanner. Selected human sequences are likely to differ in sequence andmake alternative contacts with the antigen from those of the originalbinding site. However, constraints imposed by binding of the portion oforiginal sequence to antigen and shapes of the antigen and its antigenbinding sites, are likely to drive new contacts of human sequences tothe same region or epitope of the antigen. This process has thereforebeen termed “epitope imprinted selection,” or “EIS.”

Starting with an animal antibody, one process results in the selectionof antibodies that are partly human antibodies. Such antibodies can besufficiently similar in sequence to human antibodies to be used directlyin therapy or after alteration of a few key residues. In EIS,repertoires of antibody fragments can be displayed on the surface offilamentous phase and genes encoding fragments with antigen bindingactivities selected by binding of the phage to antigen.

Yet additional methods for humanizing antibodies are described in U.S.Pat. Nos. 5,750,078; 5,502,167; 5,705,154; 5,770,403; 5,698,417;5,693,493; 5,558,864; 4,935,496; and 4,816,567, each incorporated hereinby reference.

As discussed in the above techniques, the advent of methods of molecularbiology and recombinant technology, it is now possible to produceantibodies by recombinant means and thereby generate gene sequences thatcode for specific amino acid sequences found in the polypeptidestructure of antibodies. This has permitted the ready production ofantibodies having sequences characteristic of inhibitory antibodies fromdifferent species and sources, as discussed above. In accordance withthe foregoing, the antibodies useful in the methods described herein areanti-neuraminidase antibodies, specifically antibodies whose specificityis toward the same epitope of neuraminidase as 2B9 and include alltherapeutically active variants and antigen binding fragments thereofwhether produced by recombinant methods or by direct synthesis of theantibody polypeptides.

As described below, the 2B9 anti-N1NA monoclonal antibody was humanized.Two humanized heavy chain sequences (designated “G2” and “G5” herein)are set forth in SEQ ID NOS:9 and 10, respectively. Two humanized lightchain sequences (designated “K3” and “K4” herein) are set forth in SEQID NOS:11 and 12, respectively.

In some embodiments, an antibody containing variant amino acid sequenceswith respect to humanized SEQ ID NOS:9-12 can be produced and used inthe compositions and methods described herein. In some cases, forexample, an antibody as provided herein can include a light chain or aheavy chain having an amino acid sequence with at least 85% (e.g., 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%,98.5%, 99.0%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequenceidentity to the heavy and light chain sequences set forth in SEQ IDNOS:9-12. Thus, this document provides antibodies that bindneuraminidase and that have the ability to inhibit neuraminidase enzymeactivity, and wherein the antibody comprises a light chain amino acidsequence that is at least 85 percent (e.g., at least 90 percent, atleast 95 percent, at least 96 percent, at least 97 percent, at least 98percent, or at least 99 percent) identical to the amino acid sequenceset forth in SEQ ID NO:9 or SEQ ID NO:10, and a heavy chain amino acidsequence that is at least 85 percent (e.g., at least 90 percent, atleast 95 percent, at least 98 percent, or at least 99 percent) identicalto the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:8.

Percent sequence identity is calculated by determining the number ofmatched positions in aligned amino acid sequences, dividing the numberof matched positions by the total number of aligned amino acids, andmultiplying by 100. A matched position refers to a position in whichidentical amino acids occur at the same position in aligned amino acidsequences. Percent sequence identity also can be determined for anynucleic acid sequence.

Percent sequence identity is determined by comparing a target nucleicacid or amino acid sequence to the identified nucleic acid or amino acidsequence using the BLAST 2 Sequences (Bl2seq) program from thestand-alone version of BLASTZ containing BLASTN version 2.0.14 andBLASTP version 2.0.14. This stand-alone version of BLASTZ can beobtained on the World Wide Web from Fish & Richardson's web site (“fr‘dot’ com ‘slash’ blast”) or the U.S. government's National Center forBiotechnology Information web site (“ncbi ‘dot’ nlm ‘dot’ nih ‘dot’gov”). Instructions explaining how to use the Bl2seq program can befound in the readme file accompanying BLASTZ.

Bl2seq performs a comparison between two sequences using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. Tocompare two nucleic acid sequences, the options are set as follows: -iis set to a file containing the first nucleic acid sequence to becompared (e.g., C:\seq1.txt); -j is set to a file containing the secondnucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set toblastn; -o is set to any desired file name (e.g., C:\output.txt); -q isset to −1; -r is set to 2; and all other options are left at theirdefault setting. The following command will generate an output filecontaining a comparison between two sequences: C:\B12seq c:\seq1.txt -jc:\seq2.txt -p blastn -o c:\output.txt -q −1-r 2. If the target sequenceshares homology with any portion of the identified sequence, thedesignated output file will present those regions of homology as alignedsequences. If the target sequence does not share homology with anyportion of the identified sequence, the designated output file will notpresent aligned sequences.

Once aligned, a length is determined by counting the number ofconsecutive nucleotides from the target sequence presented in alignmentwith sequence from the identified sequence starting with any matchedposition and ending with any other matched position. A matched positionis any position where an identical nucleotide is presented in both thetarget and identified sequence. Gaps presented in the target sequenceare not counted since gaps are not nucleotides. Likewise, gaps presentedin the identified sequence are not counted since target sequencenucleotides are counted, not nucleotides from the identified sequence.

The percent identity over a particular length is determined by countingthe number of matched positions over that length and dividing thatnumber by the length followed by multiplying the resulting value by 100.For example, if (1) a target sequence that is 450 amino acids in lengthis compared to the sequence set forth in SEQ ID NO:7, (2) the Bl2seqprogram presents 447 amino acids from the target sequence aligned with aregion of the sequence set forth in SEQ ID NO:7 where the first and lastamino acids of that 447 amino acid region are matches, and (3) thenumber of matches over those 447 aligned amino acids is 445, then the450 amino acid target sequence contains a length of 447 and a percentidentity over that length of (i.e., 445) 447×100=99.6%).

The percent identity over the full length of an amino acid sequence isdetermined by counting the number of matched positions over the entirelength of the query sequence (e.g., SEQ ID NO:7), dividing that numberby the length of the query sequence, and multiplying by 100. Forexample, if the Bl2seq program presents 447 amino acids from the 450amino acid target sequence aligned with and matching the 464 amino acidsin the SEQ ID NO:7 query sequence, then the 450 amino acid targetsequence is 96.3 percent identical to SEQ ID NO:7 (447/464=96.3%).

It will be appreciated that different regions within a single amino acidor nucleic acid target sequence that aligns with an identified sequencecan each have their own percent identity. It is noted that the percentidentity value is rounded to the nearest tenth. For example, 78.11,78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16,78.17, 78.18, and 78.19 are rounded up to 78.2. It also is noted thatthe length value will always be an integer.

Variant antibodies having one or more amino acid substitution relativeto the amino acid sequences set forth in SEQ DI NOS:9-12, for example,can be prepared and modified as described herein Amino acidsubstitutions can be made, in some cases, by selecting substitutionsthat do not differ significantly in their effect on maintaining (a) thestructure of the peptide backbone in the area of the substitution, (b)the charge or hydrophobicity of the molecule at the target site, or (c)the bulk of the side chain. For example, naturally occurring residuescan be divided into groups based on side-chain properties: (1)hydrophobic amino acids (norleucine, methionine, alanine, valine,leucine, and isoleucine); (2) neutral hydrophilic amino acids (cysteine,serine, and threonine); (3) acidic amino acids (aspartic acid andglutamic acid); (4) basic amino acids (asparagine, glutamine, histidine,lysine, and arginine); (5) amino acids that influence chain orientation(glycine and proline); and (6) aromatic amino acids (tryptophan,tyrosine, and phenylalanine). Substitutions made within these groups canbe considered conservative substitutions. Non-limiting examples ofuseful substitutions include, without limitation, substitution of valinefor alanine, lysine for arginine, glutamine for asparagine, glutamicacid for aspartic acid, serine for cysteine, asparagine for glutamine,aspartic acid for glutamic acid, proline for glycine, arginine forhistidine, leucine for isoleucine, isoleucine for leucine, arginine forlysine, leucine for methionine, leucine for phenyalanine, glycine forproline, threonine for serine, serine for threonine, tyrosine fortryptophan, phenylalanine for tyrosine, and/or leucine for valine.

In some embodiments, an antibody can include one or morenon-conservative substitutions. Non-conservative substitutions typicallyentail exchanging a member of one of the classes described above for amember of another class. Such production can be desirable to providelarge quantities or alternative embodiments of such compounds. Whetheran amino acid change results in a functional polypeptide can readily bedetermined by assaying the specific activity of the peptide variant.

Plant Production of Antibodies:

It is to be noted that the materials and methods described herein forexpressing antigenic polypeptides in plants also can be used to generateplant-produced antibodies (e.g., the 2B9 monoclonal antibodies describedherein). When an antibody is expressed in plants, it is to be understoodthat the heavy and light chains can be expressed from the same vector,or from two separate vectors. In some embodiments, antibody polypeptidescan be produced in a plant using an agrobacterial vector that launches aviral construct (i.e., an RNA with characteristics of a plant virus)encoding the polypeptide of interest. The RNA can have characteristicsof (and/or include sequences of), for example, TMV.

A “launch vector” typically contains agrobacterial sequences includingreplication sequences, and also contains plant viral sequences(including self-replication sequences) that carry a gene encoding apolypeptide of interest. See, e.g., Musiychuk et al. (2006) Influenzaand Other Respiratory Viruses, Blackwell Publishing Ltd, 1:19-25;incorporated herein by reference). A launch vector can be introducedinto plant tissue (e.g., by agroinfiltration), which allowssubstantially systemic delivery. For transient transformation,non-integrated T-DNA copies of the launch vector remain transientlypresent in the nucleolus and are transcribed leading to the expressionof the carrying genes (Kapila et al. (1997) Plant Science 122:101;incorporated herein by reference). Agrobacterium-mediated transientexpression, differently from viral vectors, cannot lead to the systemicspreading of the expression of the gene of interest. One advantage ofthis system is the possibility to clone genes larger than 2 kb togenerate constructs that would be impossible to obtain with viralvectors (Voinnet et al. (2003) Plant J. 33:949; incorporated herein byreference). Furthermore, using such techniques, it is possible totransform a plant with more than one transgene, such that multimericproteins (e.g., antibodies or subunits of complexed proteins) can beexpressed and assembled. Furthermore, the possibility of co-expressionof multiple transgenes by means of co-infiltration with differentAgrobacterium can be taken advantage of, either by separate infiltrationor using mixed cultures.

In some embodiments, a launch vector can include sequences that allowfor selection (or at least detection) in Agrobacteria and also forselection/detection in infiltrated tissues. Furthermore, a launch vectortypically includes sequences that are transcribed in the plant to yieldviral RNA production, followed by generation of viral proteins.Production of viral proteins and viral RNA can yield rapid production ofmultiple copies of RNA encoding the pharmaceutically active protein ofinterest. Such production can result in rapid protein production of thetarget of interest in a relatively short period of time. Thus, a highlyefficient system for protein production can be generated.

The agroinfiltration technique utilizing viral expression vectors can beused to produce limited quantity of protein of interest in order toverify the expression levels before deciding if it is worth generatingtransgenic plants. Alternatively or additionally, the agroinfiltrationtechnique utilizing viral expression vectors is useful for rapidgeneration of plants capable of producing huge amounts of protein as aprimary production platform. Thus, this transient expression system canbe used on industrial scale.

Further provided are any of a variety of different Agrobacterialplasmids, binary plasmids, or derivatives thereof such as pBIV, pBI1221,pGreen, etc., which can be used in the methods provided herein. Numeroussuitable vectors are known in the art and can be directed and/ormodified according to methods known in the art, or those describedherein so as to utilize in the methods described provided herein.

An exemplary launch vector, pBID4, contains the 35S promoter ofcauliflower mosaic virus (a DNA plant virus) that drives initialtranscription of the recombinant viral genome following introductioninto plants, and the nos terminator, the transcriptional terminator ofAgrobacterium nopaline synthase. The vector further contains sequencesof the tobacco mosaic virus genome including genes for virus replication(126/183K) and cell-t-cell movement (MP). The vector further contains agene encoding a polypeptide of interest, inserted into a unique cloningsite within the tobacco mosaic virus genome sequences and under thetranscriptional control of the coat protein subgenomic mRNA promoter.Because this “target gene” (i.e., gene encoding a protein or polypeptideof interest) replaces coding sequences for the TMV coat protein, theresultant viral vector is naked self-replicating RNA that is lesssubject to recombination than CP-containing vectors, and that cannoteffectively spread and survive in the environment. Left and right bordersequences (LB and RB) delimit the region of the launch vector that istransferred into plant cells following infiltration of plants withrecombinant Agrobacterium carrying the vector. Upon introduction ofagrobacteria carrying this vector into plant tissue (typically byagroinfiltration but alternatively by injection or other means),multiple single-stranded DNA (ssDNA) copies of sequence between LB andRB are generated and released in a matter of minutes. These introducedsequences are then amplified by viral replication. Translation of thetarget gene results in accumulation of large amounts of target proteinor polypeptide in a short period of time. A launch vector can includecoat proteins and movement protein sequences.

Once produced in a plant, any suitable method can be used to partiallyor completely isolate an expressed antibody from plant material. Asdiscussed above, a wide range of fractionation and separation proceduresare known for purifying substances from plant tissues or fluids. See,also, the methods described in Example 5 herein.

Therapeutic Compositions and Uses Thereof

The antibodies provided herein, as well as plants, plant cells, andplant tissues expressing the antibodies provided herein, can havepharmaceutical activity when administered to a subject in need thereof(e.g., a vertebrate such as a mammal, including mammals such as humans,as well as veterinary animals such as bovines, ovines, canines, andfelines). Thus, this document provides therapeutic compositionscontaining the antibodies, plants, and/or plant tissues and cellsdescribed herein. Also provided herein is the use of an antibody, plant,or portion of a plant as described herein in the manufacture of amedicament for treating or preventing influenza infection.

Treatment of a subject with an influenza antibody can elicit aphysiological effect. An antibody or antigen binding fragment thereofcan have healing curative or palliative properties against a disorder ordisease and can be administered to ameliorate relieve, alleviate, delayonset of, reverse or lessen symptoms or severity of a disease ordisorder. An antibody composition can have prophylactic properties andcan be used to prevent or delay the onset of a disease or to lessen theseverity of such disease, disorder, or pathological condition when itdoes emerge.

The pharmaceutical preparations can be administered in a wide variety ofways to a subject, such as, for example, orally, nasally, enterally,parenterally, intramuscularly or intravenously, rectally, vaginally,topically, ocularly, pulmonarily, or by contact application. In someembodiments, an anti-influenza antibody expressed in a plant, or aportion thereof, can be extracted and/or purified, and used forpreparation of a pharmaceutical composition. It may be desirable toformulate such isolated products for their intended use (e.g., as apharmaceutical agent, antibody composition, etc.). In some embodiments,it will be desirable to formulate products together with some or all ofplant tissues that express them. In cases where it is desirable toformulate product together with the plant material, it will often bedesirable to have utilized a plant that is not toxic to the relevantrecipient (e.g., a human or other animal). Relevant plant tissue (e.g.,cells, roots, leaves) can simply be harvested and processed according totechniques known in the art, with due consideration to maintainingactivity of the expressed product.

An antibody or antigen binding fragment thereof (i.e., an anti-influenzaantibody or antigen binding fragment thereof) can be formulatedaccording to known techniques. For example, an effective amount of anantibody product can be formulated together with one or more organic orinorganic, liquid or solid, pharmaceutically suitable carrier materials.An antibody or antigen binding fragment thereof can be employed indosage forms such as tablets, capsules, troches, dispersions,suspensions, solutions, gelcaps, pills, caplets, creams, ointments,aerosols, powder packets, liquid solutions, solvents, diluents, surfaceactive agents, isotonic agents, thickening or emulsifying agents,preservatives, and solid bindings, as long as the biological activity ofthe protein is not destroyed by such dosage form.

In general, compositions can comprise any of a variety of differentpharmaceutically acceptable carrier(s) or vehicle(s), or a combinationof one or more such carrier(s) or vehicle(s). As used herein thelanguage “pharmaceutically acceptable carrier, adjuvant, or vehicle”includes solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Materials that canserve as pharmaceutically acceptable carriers include, but are notlimited to sugars such as lactose, glucose and sucrose; starches such ascorn starch and potato starch; cellulose and its derivatives such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients such as cocoabutter and suppository waxes; oils such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycolssuch a propylene glycol; esters such as ethyl oleate and ethyl laurate;agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother nontoxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening agents, flavoring agents, and perfumingagents, preservatives, and antioxidants can be present in thecomposition, according to the judgment of the formulator (see alsoRemington's Pharmaceutical Sciences, Fifteenth Edition, E. W. Martin,Mack Publishing Co., Easton, Pa., 1975). For example, antibody orantigen binding fragment product can be provided as a pharmaceuticalcomposition by means of conventional mixing granulating dragee-making,dissolving, lyophilizing, or similar processes.

Additional Components:

Compositions can include additionally any suitable components to enhancethe effectiveness of the composition when administered to a subject. Incertain situations, it can be desirable to prolong the effect of anantibody or antigen binding fragment thereof by slowing the absorptionof one or more components of the antibody product (e.g., protein) thatis subcutaneously or intramuscularly injected. This can be accomplishedby use of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of product then dependsupon its rate of dissolution, which in turn, can depend upon size andform. Alternatively or additionally, delayed absorption of aparenterally administered product is accomplished by dissolving orsuspending the product in an oil vehicle. Injectable depot forms aremade by forming microcapsule matrices of protein in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofproduct to polymer and the nature of the particular polymer employed,rate of release can be controlled. Examples of biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations can be prepared by entrapping product in liposomes ormicroemulsions that are compatible with body tissues.

Enterally administered preparations of antibody can be introduced insolid, semi-solid, suspension or emulsion form and can be compoundedwith any pharmaceutically acceptable carriers, such as water, suspendingagents, and emulsifying agents. In some cases, antibodies can beadministered by means of pumps or sustained-release forms, especiallywhen administered as a preventive measure, so as to prevent thedevelopment of disease in a subject or to ameliorate or delay an alreadyestablished disease. Supplementary active compounds, e.g., compoundsindependently active against the disease or clinical condition to betreated, or compounds that enhance activity of a compound as providedherein, can be incorporated into or administered with compositions.Flavorants and coloring agents can be used.

Root lines, cell lines, plants, extractions, powders, dried preparationsand purified protein or nucleic acid products, etc., can be inencapsulated form with or without one or more excipients as noted above.Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings, release controlling coatings and other coatings well known inthe pharmaceutical formulating art. In such solid dosage forms activeagent can be mixed with at least one inert diluent such as sucrose,lactose or starch. Such dosage forms can comprise, as is normalpractice, additional substances other than inert diluents, e.g.,tableting lubricants and other tableting aids such as magnesium stearateand microcrystalline cellulose. In the case of capsules, tablets andpills, the dosage forms can comprise buffering agents. They optionallycan contain opacifying agents and can be of a composition that theyrelease the active ingredient(s) only, or substantially, in a certainpart of the intestinal tract, and/or in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes.

Pharmaceutical compositions can be administered therapeutically orprophylactically. The compositions can be used to treat or prevent adisease. For example, any individual who suffers from a disease or whois at risk of developing a disease can be treated. It will beappreciated that an individual can be considered at risk for developinga disease without having been diagnosed with any symptoms of thedisease. For example, if the individual is known to have been, or to beintended to be, in situations with relatively high risk of exposure toinfluenza infection, that individual will be considered at risk fordeveloping the disease. Similarly, if members of an individual's familyor friends have been diagnosed with influenza infection, the individualcan be considered to be at risk for developing the disease.

Compositions for rectal or vaginal administration can be suppositoriesor retention enemas, which can be prepared by mixing the compositionsprovided herein with suitable non-irritating excipients or carriers suchas cocoa butter, polyethylene glycol or a suppository wax which can besolid at ambient temperature but liquid at body temperature andtherefore melt in the rectum or vaginal cavity and release the activeprotein.

Dosage forms for topical, transmucosal or transdermal administration ofa composition include ointments, pastes, creams, lotions, gels, powders,solutions, sprays, inhalants or patches. The active agent, orpreparation thereof, is admixed under sterile conditions with apharmaceutically acceptable carrier and any needed preservatives orbuffers as can be required. For transmucosal or transdermaladministration, penetrants appropriate to the barrier to be permeatedcan be used in the formulation. Such penetrants are generally known inthe art, and include, for example, for transmucosal administration,detergents, bile salts, and fusidic acid derivatives. Transmucosaladministration can be accomplished through the use of nasal sprays orsuppositories. For transdermal administration, ointments, salves, gels,or cream formulations as generally known in the art can be used.Ophthalmic formulations, eardrops, and eye drops also are contemplated.Also contemplated is the use of transdermal patches, which have theadded advantage of providing controlled delivery of a protein to thebody. Such dosage forms can be made by suspending or dispensing theproduct in the proper medium. Absorption enhancers can be used toincrease the flux of the protein across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the protein in a polymer matrix or gel.

This document provides methods for using the antibodies and compositionsprovided herein to treat or prevent an influenza infection in a subject.Compositions can be administered in such amounts and for such time as isnecessary to achieve the desired result. In some embodiments, a“therapeutically effective amount” of a pharmaceutical composition isthat amount effective for treating, attenuating, or preventing a diseasein a subject. Thus, the “amount effective to treat, attenuate, orprevent disease,” as used herein, refers to a nontoxic but sufficientamount of the pharmaceutical composition to treat, attenuate, or preventdisease in any subject. For example, the “therapeutically effectiveamount” can be an amount to treat, attenuate, or prevent infection(e.g., viral infection, influenza infection), etc.

The exact amount required can vary from subject to subject, depending onthe species, age, and general condition of the subject, the stage of thedisease, the particular pharmaceutical mixture, its mode ofadministration, and the like. Influenza antibodies, including plantsexpressing antibodies and/or preparations thereof can be formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form,” as used herein, refers to aphysically discrete unit of composition appropriate for the patient tobe treated. It will be understood, however, that the total daily usageof the compositions typically is decided by an attending physicianwithin the scope of sound medical judgment. The specific therapeuticallyeffective dose level for any particular patient or organism can dependupon a variety of factors including the severity or risk of infection;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex of the patient, dietof the patient, pharmacokinetic condition of the patient, the time ofadministration, route of administration, and rate of excretion ordegradation of the specific antibodies employed; the duration of thetreatment; drugs used in combination or coincidental with thecomposition employed; and like factors well known in the medical arts.

It will be appreciated that the compositions provided herein can beemployed in combination therapies (e.g., combination vaccine therapies).That is, pharmaceutical compositions can be administered concurrentlywith, prior to, or subsequent to, one or more other desiredpharmaceutical and/or vaccination procedures. The particular combinationof therapies (e.g., vaccines, therapeutic treatment of influenzainfection) to employ in a combination regimen will generally take intoaccount compatibility of the desired therapeutics and/or procedures andthe desired therapeutic effect to be achieved. It will be appreciatedthat the therapies and/or vaccines employed can achieve a desired effectfor the same disorder (for example, an influenza antibody can beadministered concurrently with an antigen), or they can achievedifferent effects.

In some embodiments, a method as provided herein can include the stepsof providing a biological sample (e.g., blood, serum, urine, sputum,tissue scrapings, cerebrospinal fluid, pleural fluid, peritoneal fluid,bladder washings, oral washings, touch preps, or fine-needle aspirates)from a subject (e.g., a human or another mammal), contacting thebiological sample with an antibody as described herein (e.g., a 2B9mAb), and, if the antibody shows detectable binding to the biologicalsample, administering the antibody to the subject. In some cases, thesubject can have been diagnosed as having influenza.

As described in the Examples below, the 2B9 antibody can bind tooseltamivir-resistant influenza strains. Thus, in some embodiments, theantibodies provided herein can be particularly useful for treatingstrains of influenza that are resistant to oseltamivir. The antibodiesalso may be useful against zanamivir-resistant influenza strains.

Methods for Typing Influenza Strains

The antibodies provided herein also can be useful in methods for typinginfluenza strains. For example, the 2B9 antibody binds to NA of the N1type, but not to N2NA. See, Example 5 below. Thus, an antibody can beused at least to determine whether a particular influenza strain islikely to be an N1 strain.

Articles of Manufacture

This document also provides articles of manufacture that containanti-influenza antibodies as described herein. The articles ofmanufacture can be used for diagnostic or therapeutic purposes. In someembodiments, for example, an article can include live sproutedseedlings, clonal entity or plant producing an antibody or antigenbinding fragment thereof, or preparations, extracts, or pharmaceuticalcompositions containing antibody in one or more containers filled withoptionally one or more additional ingredients of pharmaceuticalcompositions. In some embodiments, an article of manufacture can includea therapeutic agent in addition to an anti-influenza antibody (e.g., aninfluenza vaccine) for use as a combination therapy. Optionallyassociated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceutical products, which notice reflects approval by the agency ofmanufacture, use, or sale for human administration.

Kits are provided that include therapeutic reagents. In someembodiments, an anti-influenza antibody can be provided in an injectableformulation for administration. In other embodiments, an anti-influenzaantibody can be provided in an inhalable formulation for administration.Pharmaceutical doses or instructions therefore can be provided in thekit for administration to an individual suffering from or at risk forinfluenza infection.

In some embodiments, a kit can be used for diagnosis or virus typing. Anantibody can be provided in a kit, and can be used to contact abiological sample from a subject to determine whether that subject hasinfluenza. Further, since an antibody such as 2B9 may to NA of one typebut not another type (e.g., may bind to NINA, but not to N2NA), a kitcan be used to determine whether an particular influenza virus is likelyto be of a particular strain (e.g., N1 vs. N2).

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Generation of Antigen Constructs

Generation of Antigen Sequences from Influenza Virus Neuraminidase:

Nucleotide sequences encoding neuraminidase of influenza virus typeVietnam H5N1 (NAV) were synthesized and confirmed as being correct. Thenucleotide and amino acid sequences were as follows.

NAV(N1)(nt): (SEQ ID NO: 3)GGATCCTTAATTAAAATGGGATTCGTGCTTTTCTCTCAGCTTCCTTCTTTCCTTCTTGTGTCTACTCTTCTTCTTTTCCTTGTGATTTCTCACTCTTGCCGTGCTCAAAATGTCGACCTTATGCTTCAGATTGGAAACATGATTTCTATTTGGGTGTCACACTCTATTCACACTGGAAACCAGCATCAGTCTGAGCCAATTTCTAACACTAACCTTTTGACTGAGAAGGCTGTGGCTTCTGTTAAGTTGGCTGGAAACTCTTCTCTTTGCCCTATTAACGGATGGGCTGTGTACTCTAAGGATAACTCTATTAGGATTGGATCTAAGGGAGATGTGTTCGTGATTAGGGAGCCATTCATTTCTTGCTCTCACCTTGAGTGCCGTACTTTCTTCCTTACTCAGGGTGCTCTTCTTAACGATAAGCACTCTAACGGAACTGTGAAGGATAGGTCTCCACACAGGACTCTTATGTCTTGTCCAGTTGGAGAAGCTCCATCTCCATACAACTCTAGATTCGAGTCTGTTGCTTGGAGTGCTTCTGCTTGCCATGATGGAACTTCATGGCTTACTATTGGAATTTCTGGACCAGATAACGGAGCTGTTGCTGTGCTTAAGTACAACGGAATTATTACTGATACCATCAAGTCTTGGAGGAACAACATTCTTAGGACTCAGGAGTCTGAGTGTGCTTGCGTTAACGGATCTTGCTTCACTGTGATGACTGATGGACCATCTAATGGACAGGCTTCTCACAAGATTTTCAAGATGGAGAAGGGAAAGGTTGTGAAGTCTGTGGAACTTGATGCTCCAAACTACCATTACGAGGAGTGTTCTTGCTATCCAGATGCTGGAGAGATTACTTGTGTGTGCCGTGATAATTGGCATGGATCTAACAGGCCATGGGTGTCATTCAATCAGAACCTTGAGTACCAGATTGGTTACATTTGCTCTGGAGTGTTCGGAGATAATCCAAGGCCAAACGATGGAACTGGATCTTGTGGACCAGTGTCATCTAATGGAGCTGGAGGAGTGAAGGGATTCTCTTTCAAGTACGGAAACGGAGTTTGGATTGGAAGGACTAAGTCTACTAACTCTAGGAGTGGATTCGAGATGATTTGGGACCCAAACGGATGGACTGAGACTGATTCTTCTTTCTCTGTGAAGCAGGATATTGTGGCTATTACTGATTGGAGTGGATACTCTGGATCTTTCGTTCAGCACCCAGAGCTTACTGGACTTGATTGCATTAGGCCATGCTTCTGGGTTGAACTTATTAGGGGAAGGCCAAAGGAGTCTACTATTTGGACTTCTGGATCTTCTATTTCTTTCTGCGGAGTGAATTCTGATACTGTGGGATGGTCTTGGCCAGATGGAGCTGAGCTTCCATTCACTATTGATAAGGTCGACCATCATCATCATCACCACA AGGATGAGCTTTGACTCGAG NAV(N1)(aa): (SEQ ID NO: 4)LMLQIGNMISIWVSHSIHTGNQHQSEPISNTNLLTEKAVASVKLAGNSSLCPINGWAVYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLNDKHSNGTVKDRSPHRTLMSCPVGEAPSPYNSRFESVAWSASACHDGTSWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTVMTDGPSNGQASHKIFKMEKGKVVKSVELDAPNYHYEECSCYPDAGEITCVCRDNWHGSNRPWVSFNQNLEYQIGYICSGVFGDNPRPNDGTGSCGPVSSNGAGGVKGFSFKYGNGVWIGRTKSTNSRSGFEMIWDPNGWTETDSSFSVKQDIVAITDWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPKESTIWTSGSSISFCGVNSDTVGW SWPDGAELPFTIDK

Example 2 Generation of Antigen Vectors

The NA target antigen constructs were subcloned into the viral vectorpBI-D4. pBI-D4 is a pBI121-derived binary vector in which the reportergene coding for the Escherichia coli beta-D-glucuronidase (GUS) isreplaced by a “polylinker” where, between the XbaI and SacI sites, aTMV-derived vector is inserted. pBI-D4 is a TMV-based construct in whicha foreign gene to be expressed (e.g., target antigen) replaces the coatprotein (CP) gene of TMV. The virus retains the TMV 126/183 kDa gene,the movement protein (MP) gene, and the CP subgenomic mRNA promoter(sgp), which extends into the CP open reading frame (ORF). The startcodon for CP has been mutated. The virus lacks CP and therefore cannotmove throughout the host plant via phloem. However, cell-to-cellmovement of viral infection remains functional, and the virus can moveslowly to the upper leaves in this manner. A multiple cloning site(PacI-PmeI-AgeI-XhoI) has been engineered at the end of sgp forexpression of foreign genes, and is followed by the TMV 3′non-translated region (NTR). The 35S promoter is fused at the 5′ end ofthe viral sequence, the vector sequence is positioned between the BamH1and Sac1 sites of pBI121, and the hammerhead ribozyme is placed 3′ ofthe viral sequence (Chen et al. (2003) Mol. Breed. 11:287).

These constructs were generated to include fusions of sequences encodingNA to sequences encoding the signal peptide from tobacco PR-1a protein,a 6× His tag and the ER-retention anchor sequence KDEL. For constructscontaining sequence encoding PR-NA-KDEL, the coding DNA was introducedinto pB1-D4 as PacI-XhoI fragments. NAV (NA Vietnam) was introduceddirectly as a PacI-XhoI fragment into pB1-D4. Nucleotide sequences weresubsequently verified spanning the subcloning junctions of the finalexpression constructs.

Example 3 Generation of Plants and Antigen Production

Agrobacterium Infiltration of Plants:

Agrobacterium-mediated transient expression system achieved byAgrobacterium infiltration was utilized (Turpen et al. (1993) J. Virol.Methods 42:227). Healthy leaves of Nicotiana benthamiana wereinfiltrated with A. rhizogenes containing viral vectors engineered toexpress N1NA.

The A. tumifaciens strain A4 (ATCC 43057; ATCC, Manassas, Va.) wastransformed with the constructs pB1-D4-PR-NA-KDEL and pB1-D4-PR-NA-VAC.Agrobacterium cultures were grown and induced as described (Kapila etal. (1997) Plant Sci. 122:101). A 2 ml starter-culture (picked from afresh colony) was grown overnight in YEB (5 g/l beef extract, 1 g/lyeast extract, 5 g/l peptone, 5 g/l sucrose, 2 mM MgSO₄) with 25 μg/mlkanamycin at 28° C. The starter culture was diluted 1:500 into 500 ml ofYEB with 25 μg/ml kanamycin, 10 mM 2-4(-morpholino)ethanesulfonic acid(MES) pH 5.6, 2 mM additional MgSO₄ and 20 μM acetosyringone. Thediluted culture was then grown overnight to an O.D.₆₀₀ of ˜1.7 at 28° C.The cells were centrifuged at 3,000×g for 15 minutes and re-suspended inMMA medium (MS salts, 10 mM MES pH 5.6, 20 g/l sucrose, 200 μMacetosyringone) to an O.D.₆₀₀ of 2.4, kept for 1-3 hour at roomtemperature, and used for Agrobacterium-infiltration. N. benthamianaleaves were injected with the Agrobacterium-suspension using adisposable syringe without a needle. Infiltrated leaves were harvested 6days post-infiltration. Plants were screened for the presence of targetantigen expression by immunoblot analysis. Zymogram analysis revealedthe expression of NA proteins in the N. benthamiana transgenic rootstested.

Example 4 Production of Antigen

100 mg samples of N. benthamiana infiltrated leaf material wereharvested at 4, 5, 6 and 7 days post-infection. The fresh tissue wasanalysed for protein expression right after being harvested or collectedat −80° C. for the preparation of subsequent crude plants extracts orfor fusion protein purification.

Fresh samples were resuspended in cold PBS 1× plus protease inhibitors(Roche) in a 1/3 w/v ratio (1 ml/0.3 g of tissue) and ground with apestle. The homogenates were boiled for 5 minutes in SDS gel loadingbuffer and then clarified by centrifugation for 5 minutes at 12,000 rpmat 4° C. The supernatants were transferred to fresh tubes, and 20 μl, 1μl, or dilutions thereof were separated by 12% SDS-PAGE and analyzed byWestern analysis using anti-His₆-HA mouse polyclonal antibodies.

NA expression in N. benthamiana plants infiltrated either with A.tumefaciens containing the plasmid pBID4-NA-KDEL led to a specific bandcorresponding to the molecular weight of NA-KDEL. Quantification ofNA-KDEL expressed in crude extract was made by immunoblotting both onmanually infiltrated tissues and on vacuum-infiltrated tissues.

Purification of Antigens:

Leaves from plants infiltrated with recombinant A. tumefacienscontaining the pBID4-NA-KDEL construct were ground by homogenization.Extraction buffer with “EDTA-free” protease inhibitors (Roche) and 1%Triton X-100 was used at a ratio of 3× (w/v) and rocked for 30 minutesat 4° C. Extracts were clarified by centrifugation at 9000×g for 10minutes at 4° C. Supernatants were sequentially filtered through Miracloth, centrifuged at 20,000×g for 30 minutes at 4° C., and filteredthrough a 0.45-μm filter before chromatographic purification.

The resulting extracts were cut using ammonium sulfate precipitation.Briefly, (NH₄)₂SO₄ was added to 20% saturation, incubated on ice for 1hour, and spun down at 18,000×g for 15 minutes. Pellets were discardedand (NH₄)₂SO₄ was added slowly to 60% saturation, incubated on ice for 1hour, and spun down at 18,000×g for 15 minutes. Supernatants werediscarded and the resulting pellets were resuspended in buffer,maintained on ice for 20 minutes, and centrifuged at 18,000×g for 30minutes. Supernatants were dialyzed overnight against 10,000 volumes ofwashing buffer.

His-tagged NA-KDEL proteins were purified using Ni-NTA sepharose(“Chelating Sepharose Fast Flow Column”; Amersham) at room temperatureunder gravity. The purification was performed under non-denaturingconditions. Proteins were collected as 0.5 ml fractions that wereunified, combined with 20 mM EDTA, dialyzed against 1×PBS overnight at4° C., and analyzed by SDS-PAGE. Alternatively, fractions werecollected, unified, combined with 20 mM EDTA, dialyzed against 10 mMNaH₂PO₄ overnight at 4° C., and purified by anion exchangechromatography. For NA-KDEL purification, anion exchange column QSepharose Fast Flow (Amersham Pharmacia Biosciences) was used. Samplesof the NA-KDEL affinity or ion-exchange purified proteins were separatedon 12% polyacrylamide gels followed by Coomassie staining.

After dialysis, samples were analyzed by immunoblotting using the mAbα-anti-His₆. The His-tag was maintained by the expressed proteins, andthe final concentration of the purified protein was determined usingGeneTools software from Syngene (Frederick, Md.).

Example 5 Derivation of a Murine Hybridoma Secreting Monoclonal Antibody

A 10 week-old female A/J mouse was injected intraperitoneally withcrudely-purified, plant-expressed vaccine material comprised of 50 μg offull-length N1 neuraminidase. Soluble protein was delivered in 300 μlwith no adjuvant. Identical doses were given 14 days and 24 days later.

Seventy-two hours after the second boost, 45 million spleen cells werefused with 5 million P3XAg8.653 murine myeloma cells using polyethyleneglycol. The resulting 50 million fused cells were plated at 5×10⁵ cellsper well in 10×96 well plates. HAT (hypoxanthine, aminopterin, andthymidine) selection followed 24 hours later and continued untilcolonies arose. All immunoglobulin-secreting hybridomas were subclonedby three rounds of limiting dilution in the presence of HAT.

Potential hybridomas were screened on ELISA plates for IgG specific forNIBRG-14, a reverse genetics-derived clone of A/Vietnam/1194/04 (NIBSC,Mill Hill, UK). Hybridoma 2B9 had a very high specific signal. Thespecificity of this monoclonal antibody was tested further by ELISAagainst plant-expressed antigens. Supernatant from 10⁶ cells, culturedfor 48 hours in 2.5 ml of Iscoves minimally essential mediumsupplemented with 10% fetal bovine serum, was strongly reactive againstNIBRG-14 and N1 neuraminidase, but not against N2 neuraminidase or H5hemagglutinin (FIG. 2).

Using an ELISA, the isotype and subclass of the 2B9 anti-N1 monoclonalantibody was determined to be IgG2b′κ.

Example 6 Engineering of mAb 2B9 in Plants

RT-PCR was performed on RNA purified from 2B9 hybridoma cells using aNovagen kit to determine the sequence of variable regions. Specificprimers then were designed to clone the full-length antibody light andheavy chain cDNAs. The nucleotide sequences were obtained using anautomated sequencer, and were translated to determine amino acidsequences for the light and heavy chains:

2B9 light chain sequence: (SEQ ID NO: 5)MRFPAQFLGLLLVWLTGARCDIQMTQSPASLSESVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYFAKTLAEGVPSTFSGSGSGTLFSLKINSLQPEDFGNYYCQHHYGTPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNEC 2B9 mAb heavy chain sequence: (SEQ ID NO: 6)MGWSWIFLLSVTAGVHSQVQLQQSGAELVRPGTSVKMSCKAAGYTFTNYWIGWVKQRPGHGLEWIGDIYPENDFSNYNEKFKDKATLTADTSSRTAYMQLSSLTSEDSAIYYCVRANEGWYLDVWGTGTTVSVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPTSTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVLTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKIKGIVRAPQVYILSPPPEQLSRKDVSLTCLAVGFSPEDISVEWTSNGHTEENYKNTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRHEGLHSYY LKKTISRSPGK 

Example 6 Characterization of Antibody Inhibitory Activity

For characterization of antibody activity, an assay based on therecommended WHO neuraminidase assay protocol was used, with minormodifications. For each assay, reactions were conducted in triplicateand consisted of:

-   -   a) 1 μl fresh extract prepared from plant tissue that had been        infiltrated with an expression vector encoding neuraminidase        (N1) lacking the N-terminal transmembrane domain; to prepare the        plant extracts 1 μl of buffer was used for each mg of plant        tissue.    -   b) no antibody (positive control) or a volume of monoclonal        antibody (either Ab αN1 [from hybridoma 2B9] or Ab RSV [antibody        against viral RSV F protein raised in mouse]), such that the        molar ratio of neuraminidase to antibody was 1:1, 1:10, 1:20 or        1:30        It is noted that the neuraminidase antibody and RSV F antibody        were of the same isotype (murine IgG2b). Reactions were        incubated at room temperature for 30 minutes to give the        antibodies the opportunity to recognize the plant-produced        neuraminidase. Reactions were then incubated at 37° C., an        optimum temperature for neuraminidase activity. Product (sialic        acid) accumulation was assessed colorimetrically at 549 nm using        a spectrophotometer, and quantified against sialic acid        standards.

The percentage of neuraminidase inhibition was calculated using theequation % inhibition=([PC−Tr]/PC)×100, where PC is the neuraminidaseactivity of the positive control, and Tr is the neuraminidase activityof the antibody/neuraminidase combination.

A molar comparison of the antibody's ability to inhibit viralneuraminidase is depicted in FIG. 3. Percent neuraminidase inhibition(calculated according to the equation above) is shown on the y-axis andthe molar ratio of neuraminidase to antibody (1:1, 1:10, 1:20 or 1:30)is shown on the x-axis as R1, R10, R20 or R30, respectively. Standarderrors are shown for p<0.05.

Inhibition of plant-expressed neuraminidase activity was observed in thepresence of the murine monoclonal antibody that was generated againstthis same plant-expressed neuraminidase. For comparison, the inabilityof an unrelated (RSV) antibody to inhibit the same plant producedneuraminidase also is shown (FIG. 3).

To determine whether anti-NA 2B9 is capable of recognizing N1 antigensfrom influenza strains besides the strain from which the 2B9 antigen wasoriginally derived, neuraminidase assays were performed using threedifferent H5N1 strains (A/Vietnam/1203/04, A/Hong Kong/156/97, andA/Indonesia/05), one H1N1 strain (A/New Caldonia/99), and one H3N2strain (A/Udorn/72). In these experiments, NA inhibition was measuredusing 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid, whichliberates a quantifiable fluorescent tag in response to sialidaseactivity. MDNA has absorption and fluorescence emission maxima ofapproximately 365 and 450, respectively, and signal can be detectedfluorometrically with a sensitivity as low as 10⁶ virus particles/ml(10⁴ particles total) with a broad linear range of 0-30 fold dilutionsof the virus stock. The system used amplified live virus which wasdiluted to the appropriate concentration in reaction buffer (100 mMsodium acetate, pH 6.5, 10 mM CaCl₂) and added directly to platescontaining 2-fold serial dilutions of the tested antibody. Becauseactive NA is located on the viral surface, no purification of NA proteinwas necessary to measure enzymatic activity. The antibody was 2-foldserially-diluted and aliquoted in triplicate into 384 microplate wells.Titrated virus (also diluted in reaction buffer) was added to the platewells, followed by a 30 minute incubation. MDNA was diluted to 0.2 mM inreaction buffer and added to the plate wells, and the reaction wasallowed to proceed for and additional 30 minutes. The reaction wasstopped by addition of 200 mM sodium carbonate, pH 9.5.

Titration of each cell-culture amplified virus strain was performedprior to the assay to establish the linear range of NA activitydetection. Oseltamivir carboxylate (Tamiflu®, 2 μM) was used as acontrol drug for this assay. Oseltamivir carboxylate is a specificinhibitor of influenza virus NA activity and is available from the SRIchemical respository.

Antibody dilutions and controls were run in triplicate assays (Table 1).Antibody concentrations from 1-250 μg/ml (final well volume) weretested, and oseltamivir carboxylate (2 μM final well concentration) wasincluded as a positive inhibition control. A summary of the IC₅₀ resultsfor each virus strain is presented in Table 1.

TABLE 1 NA assay IC₅₀* results Virus Antibody IC₅₀ (μg/ml) A/Udorn/72N/D** A/NC/99 125-250 A/VN/04 <1 A/HK/97 16-33 A/Indo/05 4-8 *IC₅₀ = 50%inhibitory concentration **N/D = not determined

Example 7 Inhibition of NA from Cross-Clade 2B9

The inhibitory effect of the 2B9 antibody against other influenzastrains, including strains resistant to oseltamivir, also was examinedResults are presented in Table 2. 2B9 demonstrated the highest level ofinhibition against the A/Vietnam/1203/04 virus, and inhibited NA fromisolates in different clades, including oseltamivir-resistant strains.The IC₅₀ for 2B9 was slightly higher when tested with A/HongKong/156/97,suggesting that 2B9 may inhibit NA from recent H5N1 isolates moreefficiently.

TABLE 2 IC₅₀ of 2B9 against various influenza strains Virus OseltamivirIC₅₀* μg/ml H3N2  S^(§) N/D^(†) A/Udorn/72 H1N1 A/New Caledonia/20/99 S125-250 H5N1 Clade 1 A/Vietnam/1203/04 S ≦1 A/Vietnam/HN/30408/05 R0.5-1  Clade 2.1 A/Indoesia/05/05 S 4-8 Clade 2.2A/Egypt-2/14724/NAMRU3/06 R 1-2 A/Egypt/14725/NAMRU3/06 R 1-2 Clade 2.3A/Hong Kong/156/97 S 16-33 Clade 3 A/duck/Hong Kong/380.5/01 R 0.3-0.5*IC₅₀, 50% inhibitory concentration ^(§)S, oseltamivir sensitive; R,oseltamivir resistant ^(†)N/D, not determined

Example 8 Mouse Challenge with a/Vn/1203/04

Mice were administered 500 ng of ascites purified 2B9 intravenously for5 days beginning at 1 hour before challenge with A/VN/1203/04. PBS wasused as a control. As shown in FIG. 4, no mice treated with PBS survivedmore than 8 days, whereas about half of the mice treated with 2B9 werestill alive at the 2-week endpoint of the study.

Example 9 Humanized 2B9

The 2B9 heavy and light chain sequences were directly subcloned into apBI121-based vector, one each for heavy (HC) and light chain (LC).Humanized sequences were obtained from Antitope Ltd. (Cambridge, UK).Sequences were optimized for plant expression by GeneArt, Inc.(Burlingame, Calif.) before being cloned into the pBI121 expressionvector.

For transient expression of the 2B9 mAb in plants, Agrobacteriumtumefaciens strain GV3101 was transformed with appropriate vectors.Bacterial cultures were grown overnight, diluted to OD₆₀₀=0.5, mixed ata ratio of 2:1 (HC:LC), and used in agroinfiltration of Nicotianabenthamiana leaves. The leaves were harvested 5 days post-infiltration.

To purify the plant-produced mAb, plant tissue was homogenized in 3volumes of extraction buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA)containing 10 mM sodium diethyldithiocarbamate, 0.5% Triton X-100 andcentrifuged for 30 minutes at 15,000×g at 4° C. The supernatant wasfiltered through Miracloth and spun at 75,000×g for 30 minutes, followedby microfiltration through 0.2 μm syringe filters. The antibodies werepurified using 5 ml HiTrap MabSelect SuRe column (GE Healthcare,Piscataway, N.J.). The antibody concentration was estimated on Coomassiestained 10% SDS-PAGE gel using whole human IgG (Jackson ImmunoresearchLaboratories, West Grove, Pa.) as a standard.

Two humanized light chains and two humanized heavy chains weregenerated, and were produced in each possible combination, as shown inthe gel depicted in FIG. 5. The sequences of the humanized light andheavy chains are shown below.

h2B9 heavy chain (G2): (SEQ ID NO: 7)MGWSLILLFLVAVATRVHSQVQLVQSGSELKKPGASVKMSCKAAGYTFTNYWIGWVRQAPGQGLEWIGDIYPENDFSNYNEKFKDRATLTADTSTRTAYMELSSLRSEDTAVYYCVRANEGWYLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLGKh2B9 heavy chain (G5): (SEQ ID NO: 8)MGWSLILLFLVAVATRVHSQVQLVQSGSELKKPGASVKVSCKAAGYTFTNYWIGWVRQAPGQGLEWIGDIYPENDFSNYNEKFKDRVTITADTSTSTAYMELSSLRSEDTAVYYCVRANEGWYLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLGK h2B9 light chain (K3): (SEQ ID NO: 9)MRVPAQLLGLLLLWLPGARCDIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLVYFAKTLAEGVPSRFSGSGSGTEFTLTISSLQPDDFANYYCQHHYGTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC h2B9 light chain (K4): (SEQ ID NO: 10)MRVPAQLLGLLLLWLPGARCDIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLVYFAKTLAEGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHHYGTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 

In addition, humanized antibodies were generated in which the heavychain glycosylation sites were mutated. These are designated as “G2M”and “G5M” in FIG. 5, for example.

The various humanized 2B9 antibodies were produced in plants andexamined by ELISA for antigen binding. As shown in FIG. 6, antibodyconcentration was correlated directly with antigen binding, although thedifferent combinations of heavy and light humanized chains had somewhatvarying antigen binding capabilities. For example, at the lowestconcentration studied, antibodies with a mutated glycosylation site intheir heavy chain demonstrated greater antigen binding than antibodieswithout a mutated heavy chain glycosylation site, with the exception ofthe G5K4 combination.

Experiments also were conducted to measure inhibition of NA activity byhumanized 2B9 antibodies produced in plants or in CHO cells. The datapresented in Table 3 show the IC₅₀ values for the various h2B9antibodies, as indicated. The IC₅₀ of 2B9 from mouse ascites was 0.82(±0.09) μg/ml.

TABLE 3 Inhibition of NA activity by plant-produced h2B9 CHO- Plant-Plant- produced produced produced humanized IC₅₀ humanized IC₅₀humanized IC₅₀ 2B9 (μg/ml) 2B9 (μg/ml) 2B9 (μg/ml) VH2VK3 2.28 G2K3 0.50(±0.09) G2MK3 0.51 (±0.3) VH2VK4 0.42 (±0.16) G2K4 1.16 (±0.17) G2MK40.77 (±0.07) VH5VK3 0.72 (±0.02) G5K3 0.45 (±0.12) G5MK3 0.54 (±0.05)VH5VK4 0.53 (±0.19) G5K4 0.21 (±0.01) G5MK4 0.35 (±0.06)

The half life of hybridoma- or plant-produced 2B9 was examined byinjecting the antibody into mice either intramuscularly orintravenously. Hybridoma- and plant-produced 2B9 had a half life of 4.3days when administered intramuscularly (FIG. 7). The half life of theantibody was less when it was administered intravenously, at 3.3 daysfor the hybridoma-produced version and 2.2 days for the plantpreparation.

Taken together, the data presented herein demonstrate that theN1NA-specific monoclonal antibody, 2B9, has broad cross-reactivity tostrains of influenza from clades 1, 2, and 3, including drug-resistantstrains. 2B9 also provides protection against homologous virus challengein vivo, and it may be useful for diagnostics and for post- andpre-exposure treatment for influenza. The plant-produced h2B9 antibodyhad IC₅₀ values similar to that of hybridoma-produced 2B9.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims. All references cited herein are incorporated byreference in their entirety.

What is claimed is:
 1. A method for treating an influenza infection in asubject in need thereof, comprising administering to the subject anamount a composition that is effective to reduce symptoms of theinfluenza infection in the subject, wherein the composition comprises apharmaceutically acceptable carrier and an antibody that bindsneuraminidase, wherein the antibody has the ability to inhibitneuraminidase enzyme activity, and wherein the antibody comprises thelight chain amino acid sequence set forth in SEQ ID NO:9 or SEQ IDNO:10, and wherein the antibody comprises the heavy chain amino acidsequence set forth in SEQ ID NO:7 or SEQ ID NO:8.
 2. The method of claim1, wherein the subject is a human patient.
 3. The method of claim 2,wherein the human patient is diagnosed as having influenza.
 4. Themethod of claim 3, wherein the human patient is diagnosed as having anoseltamivir-resistant strain of influenza.